<?xml version="1.0" encoding="US-ASCII" standalone="no"?>
<!--Generated by ThunderDome XBRL - 08:26PM UTC 2023-06-28--><link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" xlink:type="simple"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" xlink:type="simple"/>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-" xlink:href="vtgn-20230331.xsd#statement-note-16-supplemental-financial-information-unaudited-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_QuarterlyFinancialInformationTextBlock" xlink:label="us-gaap_QuarterlyFinancialInformationTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_QuarterlyFinancialInformationTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-document-and-entity-information" xlink:href="vtgn-20230331.xsd#statement-document-and-entity-information" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-document-and-entity-information" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorFirmId" xlink:label="dei_AuditorFirmId" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorLocation" xlink:label="dei_AuditorLocation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_AuditorName" xlink:label="dei_AuditorName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentAnnualReport" xlink:label="dei_DocumentAnnualReport" xlink:type="locator"/>
	<link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFinStmtErrorCorrectionFlag" xlink:label="dei_DocumentFinStmtErrorCorrectionFlag" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentInformationLineItems" xlink:label="dei_DocumentInformationLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentInformationTable" xlink:label="dei_DocumentInformationTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_DocumentType" xlink:label="dei_DocumentType" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityAddressStateOrProvince" xlink:label="dei_EntityAddressStateOrProvince" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityPublicFloat" xlink:label="dei_EntityPublicFloat" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityVoluntaryFilers" xlink:label="dei_EntityVoluntaryFilers" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityWellKnownSeasonedIssuer" xlink:label="dei_EntityWellKnownSeasonedIssuer" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_IcfrAuditorAttestationFlag" xlink:label="dei_IcfrAuditorAttestationFlag" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="dei_DocumentInformationTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentInformationTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCentralIndexKey" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityRegistrantName" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AmendmentFlag" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CurrentFiscalYearEndDate" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFiscalYearFocus" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentType" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentAnnualReport" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentPeriodEndDate" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentTransitionReport" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFileNumber" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityTaxIdentificationNumber" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressAddressLine1" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressCityOrTown" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressStateOrProvince" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityAddressPostalZipCode" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_CityAreaCode" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_LocalPhoneNumber" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_Security12bTitle" xlink:type="arc"/>
    <link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_TradingSymbol" xlink:type="arc"/>
    <link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_SecurityExchangeName" xlink:type="arc"/>
    <link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityWellKnownSeasonedIssuer" xlink:type="arc"/>
    <link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityVoluntaryFilers" xlink:type="arc"/>
    <link:definitionArc order="25" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCurrentReportingStatus" xlink:type="arc"/>
    <link:definitionArc order="26" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityInteractiveDataCurrent" xlink:type="arc"/>
    <link:definitionArc order="27" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityFilerCategory" xlink:type="arc"/>
    <link:definitionArc order="28" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntitySmallBusiness" xlink:type="arc"/>
    <link:definitionArc order="29" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityEmergingGrowthCompany" xlink:type="arc"/>
    <link:definitionArc order="30" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_IcfrAuditorAttestationFlag" xlink:type="arc"/>
	<link:definitionArc order="31" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_DocumentFinStmtErrorCorrectionFlag" xlink:type="arc"/>
    <link:definitionArc order="32" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityShellCompany" xlink:type="arc"/>
    <link:definitionArc order="33" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityPublicFloat" xlink:type="arc"/>
    <link:definitionArc order="34" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="35" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorName" xlink:type="arc"/>
    <link:definitionArc order="36" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorLocation" xlink:type="arc"/>
    <link:definitionArc order="37" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="dei_DocumentInformationLineItems" xlink:to="dei_AuditorFirmId" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-consolidated-balance-sheets-parentheticals" xlink:href="vtgn-20230331.xsd#statement-consolidated-balance-sheets-parentheticals" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-consolidated-balance-sheets-parentheticals" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesIssued" xlink:label="us-gaap_CommonStockSharesIssued" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TreasuryStockCommonShares" xlink:label="us-gaap_TreasuryStockCommonShares" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TreasuryStockCommonShares" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-1-description-of-business" xlink:href="vtgn-20230331.xsd#statement-note-1-description-of-business" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-1-description-of-business" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionAxis" xlink:label="us-gaap_AssetAcquisitionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:label="us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PherinAcquisitionMember" xlink:label="vtgn_PherinAcquisitionMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AssetAcquisitionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionDomain" xlink:to="vtgn_PherinAcquisitionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_BusinessDescriptionAndBasisOfPresentationTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-2-basis-of-presentation-and-going-concern" xlink:href="vtgn-20230331.xsd#statement-note-2-basis-of-presentation-and-going-concern" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-2-basis-of-presentation-and-going-concern" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BasisOfAccounting" xlink:label="us-gaap_BasisOfAccounting" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeAxis" xlink:label="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ATMFacilityMember" xlink:label="vtgn_ATMFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JefferiesLLCMember" xlink:label="vtgn_JefferiesLLCMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ReverseStockSplitMember" xlink:label="vtgn_ReverseStockSplitMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonmonetaryTransactionTypeDomain" xlink:to="vtgn_ReverseStockSplitMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_ATMFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_JefferiesLLCMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_BasisOfAccounting" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies" xlink:href="vtgn-20230331.xsd#statement-note-3-summary-of-significant-accounting-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ManufacturingFacilityMember" xlink:label="us-gaap_ManufacturingFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MoneyMarketFundsMember" xlink:label="us-gaap_MoneyMarketFundsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedStockMember" xlink:label="us-gaap_RestrictedStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SignificantAccountingPoliciesTextBlock" xlink:label="us-gaap_SignificantAccountingPoliciesTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TechnologyEquipmentMember" xlink:label="us-gaap_TechnologyEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:label="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:label="vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LincolnParkCapitalFundMember" xlink:label="vtgn_LincolnParkCapitalFundMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_TechnologyEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ManufacturingFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_LincolnParkCapitalFundMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_MoneyMarketFundsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SignificantAccountingPoliciesTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets" xlink:href="vtgn-20230331.xsd#statement-note-4-prepaid-expenses-and-other-current-assets" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PrepaidExpensesAndOtherCurrentAssetsDisclosureTextBlock" xlink:label="vtgn_PrepaidExpensesAndOtherCurrentAssetsDisclosureTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="vtgn_PrepaidExpensesAndOtherCurrentAssetsDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-net" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermAxis" xlink:label="us-gaap_LeaseContractualTermAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentDisclosureTextBlock" xlink:label="us-gaap_PropertyPlantAndEquipmentDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:label="vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LeaseContractualTermAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses" xlink:href="vtgn-20230331.xsd#statement-note-6-accrued-expenses" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:label="us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_TerminatedFormerEmployeeMember" xlink:label="vtgn_TerminatedFormerEmployeeMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_TitleOfIndividualAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="vtgn_TerminatedFormerEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_AccountsPayableAndAccruedLiabilitiesDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-" xlink:href="vtgn-20230331.xsd#statement-note-7-note-payable-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtDisclosureTextBlock" xlink:label="us-gaap_DebtDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PromissoryNoteMember" xlink:label="vtgn_PromissoryNoteMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="vtgn_PromissoryNoteMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DebtDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:label="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesAPreferredStockMember" xlink:label="us-gaap_SeriesAPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="us-gaap_SeriesCPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesDPreferredStockMember" xlink:label="us-gaap_SeriesDPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:label="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ATMFacilityMember" xlink:label="vtgn_ATMFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Amended2019PlanMember" xlink:label="vtgn_Amended2019PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightModifiedMember" xlink:label="vtgn_ClassOfWarrantOrRightModifiedMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:label="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JefferiesLLCMember" xlink:label="vtgn_JefferiesLLCMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2019EmployeeStockPurchasePlanMember" xlink:label="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2020DecemberPublicOfferingMember" xlink:label="vtgn_The2020DecemberPublicOfferingMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_TheWinter2019WarrantModificationMember" xlink:label="vtgn_TheWinter2019WarrantModificationMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesAPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesCPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesDPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_The2020DecemberPublicOfferingMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_ATMFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_JefferiesLLCMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Amended2019PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_ClassOfWarrantOrRightModifiedMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_TheWinter2019WarrantModificationMember" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementLocationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_StockholdersEquityNoteDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-9-research-and-development-expenses-" xlink:href="vtgn-20230331.xsd#statement-note-9-research-and-development-expenses-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-9-research-and-development-expenses-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionAxis" xlink:label="us-gaap_AssetAcquisitionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchDevelopmentAndComputerSoftwareDisclosureTextBlock" xlink:label="us-gaap_ResearchDevelopmentAndComputerSoftwareDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PherinAcquisitionMember" xlink:label="vtgn_PherinAcquisitionMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AssetAcquisitionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionDomain" xlink:to="vtgn_PherinAcquisitionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_ResearchDevelopmentAndComputerSoftwareDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DomesticCountryMember" xlink:label="us-gaap_DomesticCountryMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxDisclosureTextBlock" xlink:label="us-gaap_IncomeTaxDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchMember" xlink:label="us-gaap_ResearchMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StateAndLocalJurisdictionMember" xlink:label="us-gaap_StateAndLocalJurisdictionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAxis" xlink:label="us-gaap_TaxCreditCarryforwardAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_DomesticCountryMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_StateAndLocalJurisdictionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxCreditCarryforwardAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxCreditCarryforwardNameDomain" xlink:to="us-gaap_ResearchMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_IncomeTaxDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements" xlink:href="vtgn-20230331.xsd#statement-note-11-licensing-sublicensing-and-collaborative-agreements" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:label="us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:label="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:label="vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:label="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_CollaborativeArrangementDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:label="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PerformanceSharesMember" xlink:label="us-gaap_PerformanceSharesMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Amended2019PlanMember" xlink:label="vtgn_Amended2019PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultantsMember" xlink:label="vtgn_ConsultantsMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EmployeeStockPurchasePlan2019Member" xlink:label="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_IndependentMembersOfTheBoardMember" xlink:label="vtgn_IndependentMembersOfTheBoardMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NewIndependentMembersOfTheBoardMember" xlink:label="vtgn_NewIndependentMembersOfTheBoardMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NewlyHiredEmployeeMember" xlink:label="vtgn_NewlyHiredEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OfficersAndEmployeesMember" xlink:label="vtgn_OfficersAndEmployeesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Plan2019Member" xlink:label="vtgn_Plan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2016EquityIncentivePlanMember" xlink:label="vtgn_The2016EquityIncentivePlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:label="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2016EquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Amended2019PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_PerformanceSharesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Plan2019Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VestingAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_GranteeStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_NewlyHiredEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_ConsultantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_OfficersAndEmployeesMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_IndependentMembersOfTheBoardMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_NewIndependentMembersOfTheBoardMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_DisclosureOfCompensationRelatedCostsShareBasedPaymentsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-13-related-party-transactions" xlink:href="vtgn-20230331.xsd#statement-note-13-related-party-transactions" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-13-related-party-transactions" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyMember" xlink:label="us-gaap_RelatedPartyMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionsDisclosureTextBlock" xlink:label="us-gaap_RelatedPartyTransactionsDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AdvisoryServicesMember" xlink:label="vtgn_AdvisoryServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultingFirmMember" xlink:label="vtgn_ConsultingFirmMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultingServicesMember" xlink:label="vtgn_ConsultingServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:label="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DrMarkSmithMember" xlink:label="vtgn_DrMarkSmithMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JoanneCurleyMember" xlink:label="vtgn_JoanneCurleyMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:label="vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_i3StrategyPartnersMember" xlink:label="vtgn_i3StrategyPartnersMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_ConsultingFirmMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:to="us-gaap_RelatedPartyMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_i3StrategyPartnersMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_AdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_DrMarkSmithMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_ConsultingServicesMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_JoanneCurleyMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_RelatedPartyTransactionsDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:label="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_CommitmentsAndContingenciesDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-15-subsequent-events" xlink:href="vtgn-20230331.xsd#statement-note-15-subsequent-events" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-15-subsequent-events" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DisclosureTextBlockAbstract" xlink:label="us-gaap_DisclosureTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeAxis" xlink:label="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventsTextBlock" xlink:label="us-gaap_SubsequentEventsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Plan2019Member" xlink:label="vtgn_Plan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ReverseStockSplitMember" xlink:label="vtgn_ReverseStockSplitMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SalesAgreementMember" xlink:label="vtgn_SalesAgreementMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DisclosureTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonmonetaryTransactionTypeDomain" xlink:to="vtgn_ReverseStockSplitMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_SalesAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Plan2019Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VestingAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DisclosureTextBlockAbstract" xlink:to="us-gaap_SubsequentEventsTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-significant-accounting-policies-policies" xlink:href="vtgn-20230331.xsd#statement-significant-accounting-policies-policies" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-significant-accounting-policies-policies" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountingPoliciesAbstract" xlink:label="us-gaap_AccountingPoliciesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:label="us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CompensationRelatedCostsPolicyTextBlock" xlink:label="us-gaap_CompensationRelatedCostsPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ComprehensiveIncomePolicyPolicyTextBlock" xlink:label="us-gaap_ComprehensiveIncomePolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConcentrationRiskCreditRisk" xlink:label="us-gaap_ConcentrationRiskCreditRisk" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerSharePolicyTextBlock" xlink:label="us-gaap_EarningsPerSharePolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementPolicyPolicyTextBlock" xlink:label="us-gaap_FairValueMeasurementPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:label="us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxPolicyTextBlock" xlink:label="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeLeasesPolicyTextBlock" xlink:label="us-gaap_LesseeLeasesPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:label="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PolicyTextBlockAbstract" xlink:label="us-gaap_PolicyTextBlockAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:label="us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchDevelopmentAndComputerSoftwarePolicyTextBlock" xlink:label="us-gaap_ResearchDevelopmentAndComputerSoftwarePolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueRecognitionPolicyTextBlock" xlink:label="us-gaap_RevenueRecognitionPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityPolicyTextBlock" xlink:label="us-gaap_StockholdersEquityPolicyTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UseOfEstimates" xlink:label="us-gaap_UseOfEstimates" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_WarrantsIssuedInConnectionWithEquityFinancingPolicyTextBlock" xlink:label="vtgn_WarrantsIssuedInConnectionWithEquityFinancingPolicyTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PolicyTextBlockAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PolicyTextBlockAbstract" xlink:to="us-gaap_AccountingPoliciesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_UseOfEstimates" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_CashAndCashEquivalentsPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ImpairmentOrDisposalOfLongLivedAssetsPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_StockholdersEquityPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_RevenueRecognitionPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ResearchDevelopmentAndComputerSoftwarePolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_CompensationRelatedCostsPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_IncomeTaxPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_LesseeLeasesPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ConcentrationRiskCreditRisk" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_FairValueMeasurementPolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="vtgn_WarrantsIssuedInConnectionWithEquityFinancingPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_ComprehensiveIncomePolicyPolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_EarningsPerSharePolicyTextBlock" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AccountingPoliciesAbstract" xlink:to="us-gaap_NewAccountingPronouncementsPolicyPolicyTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-tables" xlink:href="vtgn-20230331.xsd#statement-note-3-summary-of-significant-accounting-policies-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-tables" xlink:href="vtgn-20230331.xsd#statement-note-4-prepaid-expenses-and-other-current-assets-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredCostsCapitalizedPrepaidAndOtherAssetsDisclosureTextBlock" xlink:label="us-gaap_DeferredCostsCapitalizedPrepaidAndOtherAssetsDisclosureTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_DeferredCostsCapitalizedPrepaidAndOtherAssetsDisclosureTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-tables" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-net-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:label="us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DepreciationAndAmortizationPropertyPlantAndEquipmentTabeTextBlock" xlink:label="vtgn_DepreciationAndAmortizationPropertyPlantAndEquipmentTabeTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ScheduleOfFinanceLeasedAssetsTableTextBlock" xlink:label="vtgn_ScheduleOfFinanceLeasedAssetsTableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_PropertyPlantAndEquipmentTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="vtgn_ScheduleOfFinanceLeasedAssetsTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="vtgn_DepreciationAndAmortizationPropertyPlantAndEquipmentTabeTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-tables" xlink:href="vtgn-20230331.xsd#statement-note-6-accrued-expenses-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfAccruedLiabilitiesTableTextBlock" xlink:label="us-gaap_ScheduleOfAccruedLiabilitiesTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfAccruedLiabilitiesTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-tables" xlink:href="vtgn-20230331.xsd#statement-note-7-note-payable-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfDebtTableTextBlock" xlink:label="us-gaap_ScheduleOfDebtTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDebtTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-tables" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTableTextBlock" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfStockByClassTextBlock" xlink:label="us-gaap_ScheduleOfStockByClassTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:label="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfStockholdersEquityNoteWarrantsOrRightsTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfStockByClassTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-tables" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:label="us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:label="us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfUnrecognizedTaxBenefitsRollForwardTableTextBlock" xlink:label="us-gaap_ScheduleOfUnrecognizedTaxBenefitsRollForwardTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEffectiveIncomeTaxRateReconciliationTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfDeferredTaxAssetsAndLiabilitiesTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfUnrecognizedTaxBenefitsRollForwardTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-tables" xlink:href="vtgn-20230331.xsd#statement-note-11-licensing-sublicensing-and-collaborative-agreements-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:label="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ContractWithCustomerAssetAndLiabilityTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-tables" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:label="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfShareBasedPaymentAwardStockOptionsValuationAssumptionsTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfShareBasedCompensationStockOptionsActivityTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-tables" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityMaturityTableTextBlock" xlink:label="us-gaap_FinanceLeaseLiabilityMaturityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseCostTableTextBlock" xlink:label="us-gaap_LeaseCostTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AssetsAndLiabilitiesLesseeTableTextBlock" xlink:label="vtgn_AssetsAndLiabilitiesLesseeTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_FinanceLeaseAssetsAndLiabilitiesTableTextBock" xlink:label="vtgn_FinanceLeaseAssetsAndLiabilitiesTableTextBock" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ScheduleOfOperatingLeaserForCashFlowDisclosuresTableTextBlock" xlink:label="vtgn_ScheduleOfOperatingLeaserForCashFlowDisclosuresTableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="vtgn_FinanceLeaseAssetsAndLiabilitiesTableTextBock" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_FinanceLeaseLiabilityMaturityTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="vtgn_AssetsAndLiabilitiesLesseeTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_LeaseCostTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityMaturityTableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="vtgn_ScheduleOfOperatingLeaserForCashFlowDisclosuresTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-tables" xlink:href="vtgn-20230331.xsd#statement-note-16-supplemental-financial-information-unaudited-tables" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-tables" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ScheduleOfQuarterlyFinancialInformationTableTextBlock" xlink:label="us-gaap_ScheduleOfQuarterlyFinancialInformationTableTextBlock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TableTextBlock" xlink:label="us-gaap_TableTextBlock" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TableTextBlock" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TableTextBlock" xlink:to="us-gaap_ScheduleOfQuarterlyFinancialInformationTableTextBlock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-1-description-of-business-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-1-description-of-business-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-1-description-of-business-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionAxis" xlink:label="us-gaap_AssetAcquisitionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionConsiderationTransferredTransactionCost" xlink:label="us-gaap_AssetAcquisitionConsiderationTransferredTransactionCost" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsToAcquireProductiveAssets" xlink:label="us-gaap_PaymentsToAcquireProductiveAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AssetAcquisitionEquityInterestIssuedOrIssuableNumberOfShares" xlink:label="vtgn_AssetAcquisitionEquityInterestIssuedOrIssuableNumberOfShares" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AssetAcquisitionPercentageEligibleToReceiveStockConsideration" xlink:label="vtgn_AssetAcquisitionPercentageEligibleToReceiveStockConsideration" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AssetAcquisitionPercentageOfStockholdersPaidInCash" xlink:label="vtgn_AssetAcquisitionPercentageOfStockholdersPaidInCash" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExpenseRelatedToAssetAcquisition" xlink:label="vtgn_ExpenseRelatedToAssetAcquisition" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:label="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NumberOfSubsidiaries" xlink:label="vtgn_NumberOfSubsidiaries" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PherinAcquisitionMember" xlink:label="vtgn_PherinAcquisitionMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AssetAcquisitionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionDomain" xlink:to="vtgn_PherinAcquisitionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_AssetAcquisitionEquityInterestIssuedOrIssuableNumberOfShares" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_AssetAcquisitionPercentageEligibleToReceiveStockConsideration" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PaymentsToAcquireProductiveAssets" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_AssetAcquisitionPercentageOfStockholdersPaidInCash" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetAcquisitionConsiderationTransferredTransactionCost" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ExpenseRelatedToAssetAcquisition" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_NumberOfSubsidiaries" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-2-basis-of-presentation-and-going-concern-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-2-basis-of-presentation-and-going-concern-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-2-basis-of-presentation-and-going-concern-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeAxis" xlink:label="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromSaleOfEquitySecuritiesFvNi" xlink:label="us-gaap_ProceedsFromSaleOfEquitySecuritiesFvNi" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromStockOptionsExercised" xlink:label="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromWarrantExercises" xlink:label="us-gaap_ProceedsFromWarrantExercises" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:label="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ATMFacilityMember" xlink:label="vtgn_ATMFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:label="vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JefferiesLLCMember" xlink:label="vtgn_JefferiesLLCMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProceedsFromGovernmentResearchGrantAwards" xlink:label="vtgn_ProceedsFromGovernmentResearchGrantAwards" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProceedsFromIssuanceOfDebtAndEquitySecurities" xlink:label="vtgn_ProceedsFromIssuanceOfDebtAndEquitySecurities" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProceedsFromLicensingAndCollaborationAgreement" xlink:label="vtgn_ProceedsFromLicensingAndCollaborationAgreement" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ReverseStockSplitMember" xlink:label="vtgn_ReverseStockSplitMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodValueSettlementOfLiabilities" xlink:label="vtgn_StockIssuedDuringPeriodValueSettlementOfLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:label="vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonmonetaryTransactionTypeDomain" xlink:to="vtgn_ReverseStockSplitMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_ATMFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_JefferiesLLCMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ProceedsFromIssuanceOfDebtAndEquitySecurities" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ProceedsFromGovernmentResearchGrantAwards" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodValueSettlementOfLiabilities" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromWarrantExercises" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromSaleOfEquitySecuritiesFvNi" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ProceedsFromLicensingAndCollaborationAgreement" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharePrice" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-3-summary-of-significant-accounting-policies-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:label="us-gaap_CashAndCashEquivalentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:label="us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredOfferingCosts" xlink:label="us-gaap_DeferredOfferingCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueByMeasurementFrequencyAxis" xlink:label="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementFrequencyDomain" xlink:label="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueMeasurementsRecurringMember" xlink:label="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinancialLiabilitiesFairValueDisclosure" xlink:label="us-gaap_FinancialLiabilitiesFairValueDisclosure" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ManufacturingFacilityMember" xlink:label="us-gaap_ManufacturingFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MoneyMarketFundsMember" xlink:label="us-gaap_MoneyMarketFundsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockDividendRatePercentage" xlink:label="us-gaap_PreferredStockDividendRatePercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:label="us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RestrictedStockMember" xlink:label="us-gaap_RestrictedStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TechnologyEquipmentMember" xlink:label="us-gaap_TechnologyEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:label="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:label="vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LincolnParkCapitalFundMember" xlink:label="vtgn_LincolnParkCapitalFundMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_UpfrontLicensePayment" xlink:label="vtgn_UpfrontLicensePayment" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_TechnologyEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ManufacturingFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_LincolnParkCapitalFundMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_CommonStockPurchaseAgreementWithLincolnParkCapitalFundMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementFrequencyDomain" xlink:to="us-gaap_FairValueMeasurementsRecurringMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByMeasurementFrequencyAxis" xlink:to="us-gaap_FairValueMeasurementFrequencyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_MoneyMarketFundsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember-default" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentUsefulLife" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredOfferingCosts" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_UpfrontLicensePayment" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNonOptionEquityInstrumentsGranted" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashAndCashEquivalentsFairValueDisclosure" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinancialLiabilitiesFairValueDisclosure" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockDividendRatePercentage" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-4-prepaid-expenses-and-other-current-assets-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NumberOfContractResearchOrganizations" xlink:label="vtgn_NumberOfContractResearchOrganizations" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ReimbursementFromCollaborationPartner" xlink:label="vtgn_ReimbursementFromCollaborationPartner" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_NumberOfContractResearchOrganizations" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ReimbursementFromCollaborationPartner" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-net-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermAxis" xlink:label="us-gaap_LeaseContractualTermAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LeaseContractualTermDomain" xlink:label="us-gaap_LeaseContractualTermDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:label="vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LeaseMonthlyPayment" xlink:label="vtgn_LeaseMonthlyPayment" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_LeaseContractualTermAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="vtgn_LeaseForOfficeEquipmentFullydepreciatedMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_LeaseMonthlyPayment" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-6-accrued-expenses-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:label="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_TerminatedFormerEmployeeMember" xlink:label="vtgn_TerminatedFormerEmployeeMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_TitleOfIndividualAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="vtgn_TerminatedFormerEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-7-note-payable-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentPeriodicPayment" xlink:label="us-gaap_DebtInstrumentPeriodicPayment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PromissoryNoteMember" xlink:label="vtgn_PromissoryNoteMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="vtgn_PromissoryNoteMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentFaceAmount" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentPeriodicPayment" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-summary-of-notes-payable-details-parentheticals" xlink:href="vtgn-20230331.xsd#statement-note-7-note-payable-summary-of-notes-payable-details-parentheticals" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-summary-of-notes-payable-details-parentheticals" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PromissoryNote388Member" xlink:label="vtgn_PromissoryNote388Member" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="vtgn_PromissoryNote388Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_CounterpartyNameAxis" xlink:label="srt_CounterpartyNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RepurchaseAgreementCounterpartyNameDomain" xlink:label="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockAmountConverted1" xlink:label="us-gaap_ConversionOfStockAmountConverted1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:label="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockSharesConverted1" xlink:label="us-gaap_ConversionOfStockSharesConverted1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockSharesIssued1" xlink:label="us-gaap_ConversionOfStockSharesIssued1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConvertiblePreferredStockSharesIssuedUponConversion" xlink:label="us-gaap_ConvertiblePreferredStockSharesIssuedUponConversion" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DividendsPreferredStock" xlink:label="us-gaap_DividendsPreferredStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockConvertibleSharesIssuable" xlink:label="us-gaap_PreferredStockConvertibleSharesIssuable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockDividendRatePercentage" xlink:label="us-gaap_PreferredStockDividendRatePercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromStockOptionsExercised" xlink:label="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromWarrantExercises" xlink:label="us-gaap_ProceedsFromWarrantExercises" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesAPreferredStockMember" xlink:label="us-gaap_SeriesAPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="us-gaap_SeriesCPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesDPreferredStockMember" xlink:label="us-gaap_SeriesDPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ATMFacilityMember" xlink:label="vtgn_ATMFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Amended2019PlanMember" xlink:label="vtgn_Amended2019PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:label="vtgn_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:label="vtgn_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightModifiedMember" xlink:label="vtgn_ClassOfWarrantOrRightModifiedMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightTerm" xlink:label="vtgn_ClassOfWarrantOrRightTerm" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:label="vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightWeightedAverageExercisePrice" xlink:label="vtgn_ClassOfWarrantOrRightWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CommonStockSharesIssuable" xlink:label="vtgn_CommonStockSharesIssuable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:label="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeferredOfferingCostsNoncurrent" xlink:label="vtgn_DeferredOfferingCostsNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JefferiesLLCMember" xlink:label="vtgn_JefferiesLLCMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedNet" xlink:label="vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedNet" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage" xlink:label="vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:label="vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2019EmployeeStockPurchasePlanMember" xlink:label="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2020DecemberPublicOfferingMember" xlink:label="vtgn_The2020DecemberPublicOfferingMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_TheWinter2019WarrantModificationMember" xlink:label="vtgn_TheWinter2019WarrantModificationMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesAPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoRegisteredCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoUnregisteredCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfAccruedDividendsOnSeriesBPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesCPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesDPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_The2020DecemberPublicOfferingMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_ATMFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CounterpartyNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RepurchaseAgreementCounterpartyNameDomain" xlink:to="vtgn_JefferiesLLCMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CounterpartyNameAxis" xlink:to="srt_RepurchaseAgreementCounterpartyNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Amended2019PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_ClassOfWarrantOrRightModifiedMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_TheWinter2019WarrantModificationMember" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementLocationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_The2019EmployeeStockPurchasePlanMember" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ConvertiblePreferredStockSharesIssuedUponConversion" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockConvertibleSharesIssuable" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ConversionOfStockSharesConverted1" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ConversionOfStockSharesIssued1" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockDividendRatePercentage" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ConversionOfStockAmountConverted1" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DividendsPreferredStock" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockSalesAgreementMaximumAggregateOfferingPrice" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_DeferredOfferingCostsNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalStockIssuedIssuanceCosts" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedNet" xlink:type="arc"/>
    <link:definitionArc order="22" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="23" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="arc"/>
    <link:definitionArc order="24" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="arc"/>
    <link:definitionArc order="25" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightWarrantsExercisedNumber" xlink:type="arc"/>
    <link:definitionArc order="26" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightExpiredDuringPeriod" xlink:type="arc"/>
    <link:definitionArc order="27" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightExpiredDuringPeriodExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="28" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromWarrantExercises" xlink:type="arc"/>
    <link:definitionArc order="29" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
    <link:definitionArc order="30" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="31" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightTerm" xlink:type="arc"/>
    <link:definitionArc order="32" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="arc"/>
    <link:definitionArc order="33" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_CommonStockSharesIssuable" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-9-research-and-development-expenses-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-9-research-and-development-expenses-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-9-research-and-development-expenses-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionAxis" xlink:label="us-gaap_AssetAcquisitionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetAcquisitionDomain" xlink:label="us-gaap_AssetAcquisitionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:label="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PherinAcquisitionMember" xlink:label="vtgn_PherinAcquisitionMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ResearchAndDevelopmentExpenseNoncash" xlink:label="vtgn_ResearchAndDevelopmentExpenseNoncash" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AssetAcquisitionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetAcquisitionDomain" xlink:to="vtgn_PherinAcquisitionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AssetAcquisitionAxis" xlink:to="us-gaap_AssetAcquisitionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ResearchAndDevelopmentExpenseNoncash" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DomesticCountryMember" xlink:label="us-gaap_DomesticCountryMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityAxis" xlink:label="us-gaap_IncomeTaxAuthorityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxAuthorityDomain" xlink:label="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLossCarryforwards" xlink:label="us-gaap_OperatingLossCarryforwards" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchMember" xlink:label="us-gaap_ResearchMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StateAndLocalJurisdictionMember" xlink:label="us-gaap_StateAndLocalJurisdictionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAmount" xlink:label="us-gaap_TaxCreditCarryforwardAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardAxis" xlink:label="us-gaap_TaxCreditCarryforwardAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TaxCreditCarryforwardNameDomain" xlink:label="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefits" xlink:label="us-gaap_UnrecognizedTaxBenefits" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CapitalizedResearchAndDevelopmentCosts" xlink:label="vtgn_CapitalizedResearchAndDevelopmentCosts" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeferredTaxAssetsTaxableIncomeLimitation" xlink:label="vtgn_DeferredTaxAssetsTaxableIncomeLimitation" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OperatingLossCarryforwardsNotSubjectToExpiration" xlink:label="vtgn_OperatingLossCarryforwardsNotSubjectToExpiration" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OperatingLossCarryforwardsSubjectToExpiration" xlink:label="vtgn_OperatingLossCarryforwardsSubjectToExpiration" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeTaxAuthorityAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_DomesticCountryMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeTaxAuthorityAxis" xlink:to="us-gaap_IncomeTaxAuthorityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeTaxAuthorityDomain" xlink:to="us-gaap_StateAndLocalJurisdictionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TaxCreditCarryforwardAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TaxCreditCarryforwardNameDomain" xlink:to="us-gaap_ResearchMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TaxCreditCarryforwardAxis" xlink:to="us-gaap_TaxCreditCarryforwardNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLossCarryforwards" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_OperatingLossCarryforwardsSubjectToExpiration" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_OperatingLossCarryforwardsNotSubjectToExpiration" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_DeferredTaxAssetsTaxableIncomeLimitation" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TaxCreditCarryforwardAmount" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_CapitalizedResearchAndDevelopmentCosts" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefits" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-11-licensing-sublicensing-and-collaborative-agreements-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CapitalizedContractCostAmortization" xlink:label="us-gaap_CapitalizedContractCostAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CapitalizedContractCostGross" xlink:label="us-gaap_CapitalizedContractCostGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CapitalizedContractCostImpairmentLoss" xlink:label="us-gaap_CapitalizedContractCostImpairmentLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiability" xlink:label="us-gaap_ContractWithCustomerLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiabilityCumulativeCatchUpAdjustmentToRevenueChangeInMeasureOfProgress" xlink:label="us-gaap_ContractWithCustomerLiabilityCumulativeCatchUpAdjustmentToRevenueChangeInMeasureOfProgress" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:label="us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:label="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:label="vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:label="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_RelatedPartyTransactionReversedExpensesFromTransactionsWithRelatedParty" xlink:label="vtgn_RelatedPartyTransactionReversedExpensesFromTransactionsWithRelatedParty" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:label="vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted" xlink:label="vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SublicenseFees" xlink:label="vtgn_SublicenseFees" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_UpfrontLicensePayment" xlink:label="vtgn_UpfrontLicensePayment" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementAffaMedAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="vtgn_CollaborativeArrangementLicenseAgreementsWithPherinPharmaceuticalsIncMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_UpfrontLicensePayment" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ContractWithCustomerLiabilityCumulativeCatchUpAdjustmentToRevenueChangeInMeasureOfProgress" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ContractWithCustomerLiability" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_SublicenseFees" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueIssuedForServices" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CapitalizedContractCostGross" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_RelatedPartyTransactionReversedExpensesFromTransactionsWithRelatedParty" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CapitalizedContractCostAmortization" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CapitalizedContractCostImpairmentLoss" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_RangeMember" xlink:label="srt_RangeMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedPayrollTaxesCurrentAndNoncurrent" xlink:label="us-gaap_AccruedPayrollTaxesCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Dividends" xlink:label="us-gaap_Dividends" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusAxis" xlink:label="us-gaap_GranteeStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GranteeStatusDomain" xlink:label="us-gaap_GranteeStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PerformanceSharesMember" xlink:label="us-gaap_PerformanceSharesMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:label="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Amended2019PlanMember" xlink:label="vtgn_Amended2019PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultantsMember" xlink:label="vtgn_ConsultantsMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EmployeeStockPurchasePlan2019Member" xlink:label="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_IndependentMembersOfTheBoardMember" xlink:label="vtgn_IndependentMembersOfTheBoardMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NewIndependentMembersOfTheBoardMember" xlink:label="vtgn_NewIndependentMembersOfTheBoardMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_NewlyHiredEmployeeMember" xlink:label="vtgn_NewlyHiredEmployeeMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OfficersAndEmployeesMember" xlink:label="vtgn_OfficersAndEmployeesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Plan2019Member" xlink:label="vtgn_Plan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardLimitationOnContributionOfCompensation" xlink:label="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardLimitationOnContributionOfCompensation" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardNumberOfSharesAuthorizedTransferred" xlink:label="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardNumberOfSharesAuthorizedTransferred" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2016EquityIncentivePlanMember" xlink:label="vtgn_The2016EquityIncentivePlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:label="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2016EquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Amended2019PlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_PerformanceSharesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Plan2019Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_RangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VestingAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_GranteeStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="us-gaap_ShareBasedPaymentArrangementEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_GranteeStatusAxis" xlink:to="us-gaap_GranteeStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_NewlyHiredEmployeeMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_ConsultantsMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_OfficersAndEmployeesMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_IndependentMembersOfTheBoardMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_GranteeStatusDomain" xlink:to="vtgn_NewIndependentMembersOfTheBoardMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardNumberOfSharesAuthorizedTransferred" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_Dividends" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharePrice" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognized" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedPayrollTaxesCurrentAndNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardLimitationOnContributionOfCompensation" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesPurchasedForAward" xlink:type="arc"/>
    <link:definitionArc order="21" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-13-related-party-transactions-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-13-related-party-transactions-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-13-related-party-transactions-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsPayableAndAccruedLiabilitiesCurrent" xlink:label="us-gaap_AccountsPayableAndAccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsPayableCurrentAndNoncurrent" xlink:label="us-gaap_AccountsPayableCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingCostsAndExpenses" xlink:label="us-gaap_OperatingCostsAndExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:label="us-gaap_RelatedAndNonrelatedPartyStatusDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyDomain" xlink:label="us-gaap_RelatedPartyDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyMember" xlink:label="us-gaap_RelatedPartyMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionAmountsOfTransaction" xlink:label="us-gaap_RelatedPartyTransactionAmountsOfTransaction" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionAxis" xlink:label="us-gaap_RelatedPartyTransactionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionDomain" xlink:label="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AdvisoryServicesMember" xlink:label="vtgn_AdvisoryServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultingFirmMember" xlink:label="vtgn_ConsultingFirmMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConsultingServicesMember" xlink:label="vtgn_ConsultingServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:label="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DrMarkSmithMember" xlink:label="vtgn_DrMarkSmithMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_JoanneCurleyMember" xlink:label="vtgn_JoanneCurleyMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:label="vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_RelatedPartyTransactionMonthlyFee" xlink:label="vtgn_RelatedPartyTransactionMonthlyFee" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_i3StrategyPartnersMember" xlink:label="vtgn_i3StrategyPartnersMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_ConsultingFirmMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedPartyTransactionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_CorporateDevelopmentAndPublicRelationsAdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionAxis" xlink:to="us-gaap_RelatedPartyTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusDomain" xlink:to="us-gaap_RelatedPartyMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedAndNonrelatedPartyStatusAxis" xlink:to="us-gaap_RelatedAndNonrelatedPartyStatusDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_i3StrategyPartnersMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_AdvisoryServicesMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_DrMarkSmithMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_ConsultingServicesMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyTransactionDomain" xlink:to="vtgn_ProjectsForCertainCnsPipelineCandidatesMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="vtgn_JoanneCurleyMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingCostsAndExpenses" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RelatedPartyTransactionAmountsOfTransaction" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_RelatedPartyTransactionMonthlyFee" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccountsPayableAndAccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccountsPayableCurrentAndNoncurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseExpense" xlink:label="us-gaap_OperatingLeaseExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseExpense" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-15-subsequent-events-details-textual" xlink:href="vtgn-20230331.xsd#statement-note-15-subsequent-events-details-textual" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-15-subsequent-events-details-textual" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockSharesAuthorized" xlink:label="us-gaap_CommonStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeAxis" xlink:label="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonmonetaryTransactionTypeDomain" xlink:label="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockParOrStatedValuePerShare" xlink:label="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:label="us-gaap_PreferredStockSharesAuthorized" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharesIssuedPricePerShare" xlink:label="us-gaap_SharesIssuedPricePerShare" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:label="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventMember" xlink:label="us-gaap_SubsequentEventMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeAxis" xlink:label="us-gaap_SubsequentEventTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsequentEventTypeDomain" xlink:label="us-gaap_SubsequentEventTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Plan2019Member" xlink:label="vtgn_Plan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ReverseStockSplitMember" xlink:label="vtgn_ReverseStockSplitMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SalesAgreementMember" xlink:label="vtgn_SalesAgreementMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_NonmonetaryTransactionTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonmonetaryTransactionTypeDomain" xlink:to="vtgn_ReverseStockSplitMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_NonmonetaryTransactionTypeAxis" xlink:to="us-gaap_NonmonetaryTransactionTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsequentEventTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_SalesAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Plan2019Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_VestingAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheTwoMember" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockParOrStatedValuePerShare" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesIssuedPricePerShare" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-assumptions-of-warrant-modification-details" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock-assumptions-of-warrant-modification-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-assumptions-of-warrant-modification-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputExercisePriceMember" xlink:label="us-gaap_MeasurementInputExercisePriceMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputExpectedDividendRateMember" xlink:label="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputExpectedTermMember" xlink:label="us-gaap_MeasurementInputExpectedTermMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputPriceVolatilityMember" xlink:label="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:label="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputSharePriceMember" xlink:label="us-gaap_MeasurementInputSharePriceMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:label="us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ClassOfWarrantOrRightWeightedAverageFairValuePerShare" xlink:label="vtgn_ClassOfWarrantOrRightWeightedAverageFairValuePerShare" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PostmodificationWarrantsMember" xlink:label="vtgn_PostmodificationWarrantsMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PremodificationWarrantsMember" xlink:label="vtgn_PremodificationWarrantsMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_PremodificationWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_MeasurementInputTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputSharePriceMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_PostmodificationWarrantsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExercisePriceMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputRiskFreeInterestRateMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedTermMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputPriceVolatilityMember" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputExpectedDividendRateMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstandingMeasurementInput" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_ClassOfWarrantOrRightWeightedAverageFairValuePerShare" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-reserves-shares-details" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock-reserves-shares-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-reserves-shares-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_Amended2019PlanMember" xlink:label="vtgn_Amended2019PlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EmployeeStockPurchasePlan2019Member" xlink:label="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OpenMarketSaleAgreementMember" xlink:label="vtgn_OpenMarketSaleAgreementMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIncentivePlansMember" xlink:label="vtgn_StockIncentivePlansMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2016EquityIncentivePlanMember" xlink:label="vtgn_The2016EquityIncentivePlanMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="us-gaap_WarrantMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2016EquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_Amended2019PlanMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_EmployeeStockPurchasePlan2019Member" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_StockIncentivePlansMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_OpenMarketSaleAgreementMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-schedule-of-fair-value-of-options-granted-blackscholes-option-pricing-model-details" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-schedule-of-fair-value-of-options-granted-blackscholes-option-pricing-model-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-schedule-of-fair-value-of-options-granted-blackscholes-option-pricing-model-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PostmodifiedStockOptionPlanMember" xlink:label="vtgn_PostmodifiedStockOptionPlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PremodifiedStockOptionPlanMember" xlink:label="vtgn_PremodifiedStockOptionPlanMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionGrantDateMarketPrice" xlink:label="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionGrantDateMarketPrice" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsNumberOfOptionShares" xlink:label="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsNumberOfOptionShares" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AwardTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="vtgn_PremodifiedStockOptionPlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="vtgn_PostmodifiedStockOptionPlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharePrice" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsNumberOfOptionShares" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionGrantDateMarketPrice" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-and-contingencies-schedule-of-financing-lease-minimum-payments-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-and-contingencies-schedule-of-financing-lease-minimum-payments-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-and-contingencies-schedule-of-financing-lease-minimum-payments-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiability" xlink:label="us-gaap_FinanceLeaseLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityCurrent" xlink:label="us-gaap_FinanceLeaseLiabilityCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:label="us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDue" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearFive" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFive" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" xlink:label="us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" xlink:label="us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearTwo" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearThree" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFour" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDueYearFive" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityPaymentsDue" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityUndiscountedExcessAmount" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiability" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityCurrent" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-minimum-lease-payments-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-minimum-lease-payments-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-minimum-lease-payments-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:label="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:label="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:label="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueNextTwelveMonths" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearTwo" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearThree" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFour" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDueYearFive" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityPaymentsDue" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LesseeOperatingLeaseLiabilityUndiscountedExcessAmount" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiability" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageRemainingLeaseTerm1" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseWeightedAverageDiscountRatePercent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-cash-flows-" xlink:href="vtgn-20230331.xsd#statement-consolidated-statements-of-cash-flows-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-cash-flows-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:label="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DividendsPayableCurrentAndNoncurrent" xlink:label="us-gaap_DividendsPayableCurrentAndNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeasePrincipalPayments" xlink:label="us-gaap_FinanceLeasePrincipalPayments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:label="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInDepositsOutstanding" xlink:label="us-gaap_IncreaseDecreaseInDepositsOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:label="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInOperatingLeaseLiability" xlink:label="us-gaap_IncreaseDecreaseInOperatingLeaseLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:label="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseRightOfUseAssetAmortizationExpense" xlink:label="us-gaap_OperatingLeaseRightOfUseAssetAmortizationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PaymentsToAcquireProductiveAssets" xlink:label="us-gaap_PaymentsToAcquireProductiveAssets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RepaymentsOfNotesPayable" xlink:label="us-gaap_RepaymentsOfNotesPayable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RightOfUseAssetObtainedInExchangeForFinanceLeaseLiability" xlink:label="us-gaap_RightOfUseAssetObtainedInExchangeForFinanceLeaseLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensation" xlink:label="us-gaap_ShareBasedCompensation" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ATMFacilityMember" xlink:label="vtgn_ATMFacilityMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfStockAmountConvertedAccruedInterest" xlink:label="vtgn_ConversionOfStockAmountConvertedAccruedInterest" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DepreciationDepletionAndAmortizationExcludingAmortizationOfOperatingLeaseAssets" xlink:label="vtgn_DepreciationDepletionAndAmortizationExcludingAmortizationOfOperatingLeaseAssets" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EquityLineMember" xlink:label="vtgn_EquityLineMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExpenseRelatedToAssetAcquisition" xlink:label="vtgn_ExpenseRelatedToAssetAcquisition" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExpenseRelatedToWriteOffOfDeferredOfferingCosts" xlink:label="vtgn_ExpenseRelatedToWriteOffOfDeferredOfferingCosts" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:label="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_InsurancePremiumsSettledIssuingNotePayable" xlink:label="vtgn_InsurancePremiumsSettledIssuingNotePayable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PaymentsToAcquireLaboratoryAndOtherEquipment" xlink:label="vtgn_PaymentsToAcquireLaboratoryAndOtherEquipment" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProceedsFromIssuanceOfCommonStockAndOptionExercisesNet" xlink:label="vtgn_ProceedsFromIssuanceOfCommonStockAndOptionExercisesNet" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ProceedsFromWarrantExercisesNet" xlink:label="vtgn_ProceedsFromWarrantExercisesNet" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_ATMFacilityMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="vtgn_EquityLineMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="vtgn_DepreciationDepletionAndAmortizationExcludingAmortizationOfOperatingLeaseAssets" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="vtgn_ExpenseRelatedToAssetAcquisition" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_FairValueAdjustmentOfWarrants" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_OperatingLeaseRightOfUseAssetAmortizationExpense" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="vtgn_ExpenseRelatedToWriteOffOfDeferredOfferingCosts" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInDepositsOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInOperatingLeaseLiability" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInContractWithCustomerLiability" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayableAndAccruedLiabilities" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="vtgn_PaymentsToAcquireLaboratoryAndOtherEquipment" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquireProductiveAssets" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="vtgn_ProceedsFromIssuanceOfCommonStockAndOptionExercisesNet" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="vtgn_ProceedsFromWarrantExercisesNet" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_FinanceLeasePrincipalPayments" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_RepaymentsOfNotesPayable" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="vtgn_InsurancePremiumsSettledIssuingNotePayable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="us-gaap_RightOfUseAssetObtainedInExchangeForFinanceLeaseLiability" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="vtgn_FairValueOfCommonStockIssuedForAssetAcqusition" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="us-gaap_DividendsPayableCurrentAndNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_CashFlowNoncashInvestingAndFinancingActivitiesDisclosureAbstract" xlink:to="vtgn_ConversionOfStockAmountConvertedAccruedInterest" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-in-condensed-consolidated-balance-sheet-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-in-condensed-consolidated-balance-sheet-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-in-condensed-consolidated-balance-sheet-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseLiability" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-cost-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-cost-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-lease-cost-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseCost" xlink:label="us-gaap_OperatingLeaseCost" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeaseCost" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-activities-in-cash-flow-statements-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-activities-in-cash-flow-statements-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-operating-activities-in-cash-flow-statements-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeasePayments" xlink:label="us-gaap_OperatingLeasePayments" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingLeasePayments" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-quarterly-results-of-operations-unaudited-details" xlink:href="vtgn-20230331.xsd#statement-note-16-supplemental-financial-information-unaudited-quarterly-results-of-operations-unaudited-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-16-supplemental-financial-information-unaudited-quarterly-results-of-operations-unaudited-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductOrServiceAxis" xlink:label="srt_ProductOrServiceAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/srt/2023/elts/srt-2023.xsd#srt_ProductsAndServicesDomain" xlink:label="srt_ProductsAndServicesDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestIncomeExpenseNet" xlink:label="us-gaap_InterestIncomeExpenseNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LicenseMember" xlink:label="us-gaap_LicenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:label="us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:label="us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Revenues" xlink:label="us-gaap_Revenues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_ProductOrServiceAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_ProductsAndServicesDomain" xlink:to="us-gaap_LicenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_ProductOrServiceAxis" xlink:to="srt_ProductsAndServicesDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_Revenues" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingExpenses" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_InterestIncomeExpenseNet" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncomeTaxExpenseBenefit" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-stockholders-equity" xlink:href="vtgn-20230331.xsd#statement-consolidated-statements-of-stockholders-equity" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-stockholders-equity" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalDividendsInExcessOfRetainedEarnings" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalDividendsInExcessOfRetainedEarnings" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:label="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ConversionOfStockNameDomain" xlink:label="us-gaap_ConversionOfStockNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockMember" xlink:label="us-gaap_PreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesAPreferredStockMember" xlink:label="us-gaap_SeriesAPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesBPreferredStockMember" xlink:label="us-gaap_SeriesBPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesCPreferredStockMember" xlink:label="us-gaap_SeriesCPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SeriesDPreferredStockMember" xlink:label="us-gaap_SeriesDPreferredStockMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharesOutstanding" xlink:label="us-gaap_SharesOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesAcquisitions" xlink:label="us-gaap_StockIssuedDuringPeriodSharesAcquisitions" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueAcquisitions" xlink:label="us-gaap_StockIssuedDuringPeriodValueAcquisitions" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TreasuryStockCommonMember" xlink:label="us-gaap_TreasuryStockCommonMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AdjustmentsToAdditionalPaidInCapitalWarrantModification" xlink:label="vtgn_AdjustmentsToAdditionalPaidInCapitalWarrantModification" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:label="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:label="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedForCash" xlink:label="vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedForCash" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:label="vtgn_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodStockOptionsExercisedValue" xlink:label="vtgn_StockIssuedDuringPeriodStockOptionsExercisedValue" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_StockIssuedDuringPeriodValueWarrantsExercised" xlink:label="vtgn_StockIssuedDuringPeriodValueWarrantsExercised" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesDPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesAPreferredIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesBPreferredStockIntoCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ConversionOfStockNameDomain" xlink:to="vtgn_ConversionOfSeriesCPreferredStockToCommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_PreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesAPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesBPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesCPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesDPreferredStockMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_TreasuryStockCommonMember" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ConversionOfStockByUniqueDescriptionAxis" xlink:to="us-gaap_ConversionOfStockNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesOutstanding" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodSharesWarrantsExercised" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodValueWarrantsExercised" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueConversionOfConvertibleSecurities" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalDividendsInExcessOfRetainedEarnings" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodSharesStockOptionsExercisedForCash" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_StockIssuedDuringPeriodStockOptionsExercisedValue" xlink:type="arc"/>
    <link:definitionArc order="15" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="arc"/>
    <link:definitionArc order="16" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="arc"/>
    <link:definitionArc order="17" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="18" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_AdjustmentsToAdditionalPaidInCapitalWarrantModification" xlink:type="arc"/>
    <link:definitionArc order="19" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesAcquisitions" xlink:type="arc"/>
    <link:definitionArc order="20" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueAcquisitions" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-schedule-of-potentially-dilutive-securities-excluded-from-earnings-per-share-computation-details" xlink:href="vtgn-20230331.xsd#statement-note-3-summary-of-significant-accounting-policies-schedule-of-potentially-dilutive-securities-excluded-from-earnings-per-share-computation-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-3-summary-of-significant-accounting-policies-schedule-of-potentially-dilutive-securities-excluded-from-earnings-per-share-computation-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:label="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PlanNameAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="vtgn_The2019OmnibusEquityIncentivePlanMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_WarrantMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-schedule-of-prepaid-expenses-and-other-current-assets-details" xlink:href="vtgn-20230331.xsd#statement-note-4-prepaid-expenses-and-other-current-assets-schedule-of-prepaid-expenses-and-other-current-assets-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-4-prepaid-expenses-and-other-current-assets-schedule-of-prepaid-expenses-and-other-current-assets-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationAxis" xlink:label="us-gaap_BalanceSheetLocationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherAssetsCurrent" xlink:label="us-gaap_OtherAssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PrepaidExpensesAndOtherCurrentAssetsMember" xlink:label="us-gaap_PrepaidExpensesAndOtherCurrentAssetsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PrepaidInsurance" xlink:label="us-gaap_PrepaidInsurance" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PrepaidMaterialsAndContractServices" xlink:label="vtgn_PrepaidMaterialsAndContractServices" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PrepaidReceivableFromCollaborationPartner" xlink:label="vtgn_PrepaidReceivableFromCollaborationPartner" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PrepaidReceivableFromCroForCancelledProject" xlink:label="vtgn_PrepaidReceivableFromCroForCancelledProject" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_BalanceSheetLocationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_BalanceSheetLocationDomain" xlink:to="us-gaap_PrepaidExpensesAndOtherCurrentAssetsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_PrepaidMaterialsAndContractServices" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PrepaidInsurance" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_PrepaidReceivableFromCroForCancelledProject" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_PrepaidReceivableFromCollaborationPartner" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherAssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-schedule-of-property-and-equipment-details" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-net-schedule-of-property-and-equipment-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-schedule-of-property-and-equipment-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:label="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="us-gaap_PropertyPlantAndEquipmentGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LaboratoryEquipmentMember" xlink:label="vtgn_LaboratoryEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ManufacturingEquipmentMember" xlink:label="vtgn_ManufacturingEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OfficeFurnitureAndEquipmentMember" xlink:label="vtgn_OfficeFurnitureAndEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_TenantImprovementsMember" xlink:label="vtgn_TenantImprovementsMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_LaboratoryEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_TenantImprovementsMember" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_OfficeFurnitureAndEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_ManufacturingEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-schedule-of-assets-subject-to-financing-lease-details" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-net-schedule-of-assets-subject-to-financing-lease-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-net-schedule-of-assets-subject-to-financing-lease-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAsset" xlink:label="us-gaap_FinanceLeaseRightOfUseAsset" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OfficeFurnitureAndEquipmentMember" xlink:label="vtgn_OfficeFurnitureAndEquipmentMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_OfficeFurnitureAndEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAsset" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-depreciation-and-amortization-expense-details" xlink:href="vtgn-20230331.xsd#statement-note-5-property-and-equipment-depreciation-and-amortization-expense-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-5-property-and-equipment-depreciation-and-amortization-expense-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DepreciationAndAmortization" xlink:label="us-gaap_DepreciationAndAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_LeasedAssetsMember" xlink:label="vtgn_LeasedAssetsMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_OwnedAssetsMember" xlink:label="vtgn_OwnedAssetsMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_OwnedAssetsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="vtgn_LeasedAssetsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DepreciationAndAmortization" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-schedule-of-accrued-expenses-details" xlink:href="vtgn-20230331.xsd#statement-note-6-accrued-expenses-schedule-of-accrued-expenses-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-6-accrued-expenses-schedule-of-accrued-expenses-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedProfessionalFeesCurrent" xlink:label="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:label="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OtherAccruedLiabilitiesCurrent" xlink:label="us-gaap_OtherAccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AccruedExpensesForMaterialsAndContractualServices" xlink:label="vtgn_AccruedExpensesForMaterialsAndContractualServices" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_AccruedExpensesForMaterialsAndContractualServices" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedProfessionalFeesCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OtherAccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AccruedLiabilitiesCurrent" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-income-tax-expense-benefit-details" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-income-tax-expense-benefit-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-income-tax-expense-benefit-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:label="us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseOther" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseOther" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseResearchAndDevelopment" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseResearchAndDevelopment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseShareBasedCompensationCost" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseShareBasedCompensationCost" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationOtherReconcilingItemsPercent" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationOtherReconcilingItemsPercent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationStateAndLocalIncomeTaxes" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationStateAndLocalIncomeTaxes" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EffectiveIncomeTaxRateReconciliationExpiredNetOperatingLossCarryforward" xlink:label="vtgn_EffectiveIncomeTaxRateReconciliationExpiredNetOperatingLossCarryforward" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_EffectiveIncomeTaxRateReconciliationWarrantModifications" xlink:label="vtgn_EffectiveIncomeTaxRateReconciliationWarrantModifications" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationStateAndLocalIncomeTaxes" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_EffectiveIncomeTaxRateReconciliationWarrantModifications" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseResearchAndDevelopment" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseShareBasedCompensationCost" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationNondeductibleExpenseOther" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_EffectiveIncomeTaxRateReconciliationExpiredNetOperatingLossCarryforward" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationChangeInDeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationOtherReconcilingItemsPercent" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-deferred-tax-assets-and-liabilities-details" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-deferred-tax-assets-and-liabilities-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-deferred-tax-assets-and-liabilities-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredIncomeTaxLiabilities" xlink:label="us-gaap_DeferredIncomeTaxLiabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsDeferredIncome" xlink:label="us-gaap_DeferredTaxAssetsDeferredIncome" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsGross" xlink:label="us-gaap_DeferredTaxAssetsGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsLiabilitiesNet" xlink:label="us-gaap_DeferredTaxAssetsLiabilitiesNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsNet" xlink:label="us-gaap_DeferredTaxAssetsNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsOperatingLossCarryforwards" xlink:label="us-gaap_DeferredTaxAssetsOperatingLossCarryforwards" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsPropertyPlantAndEquipment" xlink:label="us-gaap_DeferredTaxAssetsPropertyPlantAndEquipment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:label="us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:label="us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccruals" xlink:label="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccruals" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxAssetsValuationAllowance" xlink:label="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredTaxLiabilitiesPropertyPlantAndEquipment" xlink:label="us-gaap_DeferredTaxLiabilitiesPropertyPlantAndEquipment" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeferredTaxAssetsCapitalizedResearchAndDevelopment" xlink:label="vtgn_DeferredTaxAssetsCapitalizedResearchAndDevelopment" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeferredTaxAssetsOperatingLeaseRightOfUseAssets" xlink:label="vtgn_DeferredTaxAssetsOperatingLeaseRightOfUseAssets" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsOperatingLossCarryforwards" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsPropertyPlantAndEquipment" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxCreditCarryforwardsResearch" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxDeferredExpenseCompensationAndBenefitsShareBasedCompensationCost" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_DeferredTaxAssetsOperatingLeaseRightOfUseAssets" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_DeferredTaxAssetsCapitalizedResearchAndDevelopment" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsDeferredIncome" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsTaxDeferredExpenseReservesAndAccruals" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsGross" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsValuationAllowance" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsNet" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxLiabilitiesPropertyPlantAndEquipment" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredIncomeTaxLiabilities" xlink:type="arc"/>
    <link:definitionArc order="14" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredTaxAssetsLiabilitiesNet" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-uncertain-tax-positions-details" xlink:href="vtgn-20230331.xsd#statement-note-10-income-taxes-uncertain-tax-positions-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-10-income-taxes-uncertain-tax-positions-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefits" xlink:label="us-gaap_UnrecognizedTaxBenefits" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:label="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefits" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsIncreasesResultingFromCurrentPeriodTaxPositions" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-consolidated-statement-of-operations-and-comprehensive-loss-details" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-consolidated-statement-of-operations-and-comprehensive-loss-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-consolidated-statement-of-operations-and-comprehensive-loss-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_IncomeStatementLocationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-finance-lease-obligations-details" xlink:href="vtgn-20230331.xsd#statement-note-14-commitments-contingencies-guarantees-and-indemnifications-finance-lease-obligations-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-14-commitments-contingencies-guarantees-and-indemnifications-finance-lease-obligations-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationAxis" xlink:label="us-gaap_BalanceSheetLocationAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_BalanceSheetLocationDomain" xlink:label="us-gaap_BalanceSheetLocationDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAsset" xlink:label="us-gaap_FinanceLeaseRightOfUseAsset" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:label="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OfficeEquipmentMember" xlink:label="us-gaap_OfficeEquipmentMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PropertyAndEquipmentNetMember" xlink:label="vtgn_PropertyAndEquipmentNetMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_OfficeEquipmentMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_BalanceSheetLocationAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_BalanceSheetLocationDomain" xlink:to="vtgn_PropertyAndEquipmentNetMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_BalanceSheetLocationAxis" xlink:to="us-gaap_BalanceSheetLocationDomain-default" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetBeforeAccumulatedAmortization" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAssetAccumulatedAmortization" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_FinanceLeaseRightOfUseAsset" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-schedule-of-warrants-details" xlink:href="vtgn-20230331.xsd#statement-note-8-capital-stock-schedule-of-warrants-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-8-capital-stock-schedule-of-warrants-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_WarrantsExpiringDecember92024Member" xlink:label="vtgn_WarrantsExpiringDecember92024Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_WarrantsExpiringJuly252025Member" xlink:label="vtgn_WarrantsExpiringJuly252025Member" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_WarrantsExpiringDecember92024Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="vtgn_WarrantsExpiringJuly252025Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-operations-and-comprehensive-loss-" xlink:href="vtgn-20230331.xsd#statement-consolidated-statements-of-operations-and-comprehensive-loss-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-consolidated-statements-of-operations-and-comprehensive-loss-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_EarningsPerShareBasic" xlink:label="us-gaap_EarningsPerShareBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_GeneralAndAdministrativeExpense" xlink:label="us-gaap_GeneralAndAdministrativeExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_IncomeTaxExpenseBenefit" xlink:label="us-gaap_IncomeTaxExpenseBenefit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_InterestIncomeExpenseNet" xlink:label="us-gaap_InterestIncomeExpenseNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:label="us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NonoperatingIncomeExpenseAbstract" xlink:label="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingExpenses" xlink:label="us-gaap_OperatingExpenses" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingExpensesAbstract" xlink:label="us-gaap_OperatingExpensesAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingIncomeLoss" xlink:label="us-gaap_OperatingIncomeLoss" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:label="us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ResearchAndDevelopmentExpense" xlink:label="us-gaap_ResearchAndDevelopmentExpense" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:label="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:label="vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_RevenueFromContractWithCustomerIncludingAssessedTaxDerecognized" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_OperatingExpensesAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_ResearchAndDevelopmentExpense" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_GeneralAndAdministrativeExpense" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OperatingExpenses" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OperatingExpensesAbstract" xlink:to="us-gaap_OperatingIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_InterestIncomeExpenseNet" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_IncomeTaxExpenseBenefit" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NetIncomeLoss" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_PreferredStockDividendsIncomeStatementImpact" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_NetIncomeLossAvailableToCommonStockholdersBasic" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_EarningsPerShareBasic" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NonoperatingIncomeExpenseAbstract" xlink:to="us-gaap_WeightedAverageNumberOfSharesOutstandingBasic" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-summary-of-notes-payable-details" xlink:href="vtgn-20230331.xsd#statement-note-7-note-payable-summary-of-notes-payable-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-7-note-payable-summary-of-notes-payable-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentIncreaseAccruedInterest" xlink:label="us-gaap_DebtInstrumentIncreaseAccruedInterest" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NotesPayable" xlink:label="us-gaap_NotesPayable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DebtInstrumentPrincipalBalance" xlink:label="vtgn_DebtInstrumentPrincipalBalance" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_PromissoryNote388Member" xlink:label="vtgn_PromissoryNote388Member" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_DebtInstrumentAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="vtgn_PromissoryNote388Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="vtgn_DebtInstrumentPrincipalBalance" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DebtInstrumentIncreaseAccruedInterest" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NotesPayable" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-changes-in-contract-liabilities-details" xlink:href="vtgn-20230331.xsd#statement-note-11-licensing-sublicensing-and-collaborative-agreements-changes-in-contract-liabilities-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-11-licensing-sublicensing-and-collaborative-agreements-changes-in-contract-liabilities-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ChangeInAccountingEstimateByTypeAxis" xlink:label="us-gaap_ChangeInAccountingEstimateByTypeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:label="us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:label="us-gaap_ChangeInAccountingEstimateTypeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredRevenue" xlink:label="us-gaap_DeferredRevenue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredRevenueCurrent" xlink:label="us-gaap_DeferredRevenueCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredRevenueNoncurrent" xlink:label="us-gaap_DeferredRevenueNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_AdditionsMember" xlink:label="vtgn_AdditionsMember" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeductionsMember" xlink:label="vtgn_DeductionsMember" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ChangeInAccountingEstimateByTypeAxis" xlink:to="us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ChangeInAccountingEstimateByTypeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:to="vtgn_AdditionsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ChangeInAccountingEstimateByTypeAxis" xlink:to="us-gaap_ChangeInAccountingEstimateTypeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ChangeInAccountingEstimateTypeDomain" xlink:to="vtgn_DeductionsMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredRevenueCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredRevenueNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_DeferredRevenue" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-consolidated-balance-sheets-" xlink:href="vtgn-20230331.xsd#statement-consolidated-balance-sheets-" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-consolidated-balance-sheets-" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccountsPayableCurrent" xlink:label="us-gaap_AccountsPayableCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:label="us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AdditionalPaidInCapital" xlink:label="us-gaap_AdditionalPaidInCapital" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Assets" xlink:label="us-gaap_Assets" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetsCurrent" xlink:label="us-gaap_AssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_AssetsCurrentAbstract" xlink:label="us-gaap_AssetsCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CollaborativeArrangementMember" xlink:label="us-gaap_CollaborativeArrangementMember" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommitmentsAndContingencies" xlink:label="us-gaap_CommitmentsAndContingencies" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_CommonStockValue" xlink:label="us-gaap_CommonStockValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiabilityCurrent" xlink:label="us-gaap_ContractWithCustomerLiabilityCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ContractWithCustomerLiabilityNoncurrent" xlink:label="us-gaap_ContractWithCustomerLiabilityNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredCosts" xlink:label="us-gaap_DeferredCosts" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DeferredCostsCurrent" xlink:label="us-gaap_DeferredCostsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_DepositsAssetsNoncurrent" xlink:label="us-gaap_DepositsAssetsNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityCurrent" xlink:label="us-gaap_FinanceLeaseLiabilityCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:label="us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_Liabilities" xlink:label="us-gaap_Liabilities" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesCurrent" xlink:label="us-gaap_LiabilitiesCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:label="us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesNoncurrent" xlink:label="us-gaap_LiabilitiesNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_LiabilitiesNoncurrentAbstract" xlink:label="us-gaap_LiabilitiesNoncurrentAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_NotesPayableCurrent" xlink:label="us-gaap_NotesPayableCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PreferredStockValue" xlink:label="us-gaap_PreferredStockValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockholdersEquityAbstract" xlink:label="us-gaap_StockholdersEquityAbstract" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TreasuryStockCommonValue" xlink:label="us-gaap_TreasuryStockCommonValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_TypeOfArrangementAxis" xlink:label="us-gaap_TypeOfArrangementAxis" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_DeferredOfferingCostsNoncurrent" xlink:label="vtgn_DeferredOfferingCostsNoncurrent" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_TypeOfArrangementAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="us-gaap_CollaborativeArrangementMember" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_DeferredCostsCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="vtgn_DeferredOfferingCostsNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_DeferredCosts" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_DepositsAssetsNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_Assets" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesCurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedLiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_NotesPayableCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_ContractWithCustomerLiabilityCurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityCurrent" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesCurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesNoncurrentAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_ContractWithCustomerLiabilityNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_FinanceLeaseLiabilityNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_LiabilitiesNoncurrent" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_Liabilities" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesNoncurrentAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquityAbstract" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_PreferredStockValue" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_CommonStockValue" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AdditionalPaidInCapital" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_TreasuryStockCommonValue" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquity" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-stock-option-outstanding-and-exercisable-details" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-stock-option-outstanding-and-exercisable-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-stock-option-outstanding-and-exercisable-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange1Member" xlink:label="vtgn_ExercisePriceRange1Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange2Member" xlink:label="vtgn_ExercisePriceRange2Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange3Member" xlink:label="vtgn_ExercisePriceRange3Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange4Member" xlink:label="vtgn_ExercisePriceRange4Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange5Member" xlink:label="vtgn_ExercisePriceRange5Member" xlink:type="locator"/>
    <link:loc xlink:href="vtgn-20230331.xsd#vtgn_ExercisePriceRange6Member" xlink:label="vtgn_ExercisePriceRange6Member" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange1Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain-default" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange2Member" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange3Member" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange4Member" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange5Member" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="vtgn_ExercisePriceRange6Member" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" xlink:type="arc"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-summary-of-stock-option-activity-details" xlink:href="vtgn-20230331.xsd#statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-summary-of-stock-option-activity-details" xlink:type="simple"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/20230331/role/statement-note-12-stock-option-plans-employee-stock-purchase-plan-and-401k-plan-summary-of-stock-option-activity-details" xlink:type="extended">
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_EntityDomain" xlink:label="dei_EntityDomain-default" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.sec.gov/dei/2023/dei-2023.xsd#dei_LegalEntityAxis" xlink:label="dei_LegalEntityAxis" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable" xlink:type="locator"/>
    <link:loc xlink:href="https://xbrl.fasb.org/us-gaap/2023/elts/us-gaap-2023.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="dei_LegalEntityAxis" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="dei_LegalEntityAxis" xlink:to="dei_EntityDomain-default" xlink:type="arc"/>
    <link:definitionArc order="0" xbrldt:closed="true" xbrldt:contextElement="segment" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" xlink:type="arc"/>
    <link:definitionArc order="1" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingNumber" xlink:type="arc"/>
    <link:definitionArc order="2" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="3" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross" xlink:type="arc"/>
    <link:definitionArc order="4" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="5" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="arc"/>
    <link:definitionArc order="6" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExercisesInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="7" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsForfeituresInPeriod" xlink:type="arc"/>
    <link:definitionArc order="8" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsForfeituresInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="9" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExpirationsInPeriod" xlink:type="arc"/>
    <link:definitionArc order="10" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardOptionsExpirationsInPeriodWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="11" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableNumber" xlink:type="arc"/>
    <link:definitionArc order="12" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisableWeightedAverageExercisePrice" xlink:type="arc"/>
    <link:definitionArc order="13" xbrldt:closed="true" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="arc"/>
  </link:definitionLink>
</link:linkbase>
