<?xml version="1.0"?>
<ownershipDocument>

    <schemaVersion>X0407</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2023-05-31</periodOfReport>

    <issuer>
        <issuerCik>0001053691</issuerCik>
        <issuerName>Diffusion Pharmaceuticals Inc.</issuerName>
        <issuerTradingSymbol>DFFN</issuerTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001297141</rptOwnerCik>
            <rptOwnerName>Hollingsworth Jane H</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>300 EAST MAIN ST.</rptOwnerStreet1>
            <rptOwnerStreet2>SUITE 101</rptOwnerStreet2>
            <rptOwnerCity>CHARLOTTESVILLE</rptOwnerCity>
            <rptOwnerState>VA</rptOwnerState>
            <rptOwnerZipCode>22902</rptOwnerZipCode>
            <rptOwnerStateDescription></rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>1</isDirector>
        </reportingOwnerRelationship>
    </reportingOwner>

    <aff10b5One>0</aff10b5One>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2023-05-31</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>M</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>183</value>
                </transactionShares>
                <transactionPricePerShare>
                    <footnoteId id="F1"/>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>A</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>1299</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2023-05-31</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>D</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
                <footnoteId id="F1"/>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>68</value>
                </transactionShares>
                <transactionPricePerShare>
                    <footnoteId id="F1"/>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>D</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>1231</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
    </nonDerivativeTable>

    <derivativeTable>
        <derivativeTransaction>
            <securityTitle>
                <value>Restricted Stock Unit</value>
            </securityTitle>
            <conversionOrExercisePrice>
                <footnoteId id="F1"/>
            </conversionOrExercisePrice>
            <transactionDate>
                <value>2023-05-31</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>M</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>183</value>
                </transactionShares>
                <transactionPricePerShare>
                    <value>0</value>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>D</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <exerciseDate>
                <footnoteId id="F1"/>
            </exerciseDate>
            <expirationDate>
                <footnoteId id="F1"/>
            </expirationDate>
            <underlyingSecurity>
                <underlyingSecurityTitle>
                    <value>Common Stock</value>
                </underlyingSecurityTitle>
                <underlyingSecurityShares>
                    <value>183</value>
                </underlyingSecurityShares>
            </underlyingSecurity>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>183</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </derivativeTransaction>
    </derivativeTable>

    <footnotes>
        <footnote id="F1">On May 31, 2023 (the &quot;Vesting Date&quot;), in connection with the vesting of previously awarded restricted stock units (&quot;RSUs&quot;), each RSU representing a contingent right to receive one share of the Issuer's common stock (a &quot;Share&quot;), the Reporting Person became entitled to receive, in accordance with Section 8.7 of the Issuer's 2015 Equity Incentive Plan (as amended, the &quot;Plan&quot;) and the terms of the underlying award agreement,  (i) 115 Shares and (ii) $263.84 in cash, representing the Fair Market Value (as defined in the 2015 Equity Plan) of the remaining portion of the award vested on the Vesting Date, based on the closing sale price of one Share reported by the NASDAQ Stock Market on such date of $3.88.  The remainder of award will continue to vest in tri-monthly installments, subject to the Reporting Person's continued service with the Issuer.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>/s/ William R. Elder, attorney-in-fact for the Reporting Person</signatureName>
        <signatureDate>2023-06-02</signatureDate>
    </ownerSignature>
</ownershipDocument>
