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

    <schemaVersion>X0306</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2016-12-27</periodOfReport>

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

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001663219</rptOwnerCik>
            <rptOwnerName>Giles Mark T</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>2020 AVON COURT, SUITE 4</rptOwnerStreet1>
            <rptOwnerStreet2></rptOwnerStreet2>
            <rptOwnerCity>CHARLOTTESVILLE</rptOwnerCity>
            <rptOwnerState>VA</rptOwnerState>
            <rptOwnerZipCode>22902</rptOwnerZipCode>
            <rptOwnerStateDescription></rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>1</isDirector>
        </reportingOwnerRelationship>
    </reportingOwner>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2016-12-27</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>J</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
                <footnoteId id="F1"/>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>290580</value>
                </transactionShares>
                <transactionPricePerShare>
                    <footnoteId id="F1"/>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>D</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>0</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>I</value>
                </directOrIndirectOwnership>
                <natureOfOwnership>
                    <value>By PANDA Holdings, LLC</value>
                    <footnoteId id="F2"/>
                </natureOfOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2016-12-28</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>P</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>290580</value>
                </transactionShares>
                <transactionPricePerShare>
                    <footnoteId id="F1"/>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>A</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>802689</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>I</value>
                </directOrIndirectOwnership>
                <natureOfOwnership>
                    <value>By MTG Investment Holdings, LLC</value>
                    <footnoteId id="F3"/>
                </natureOfOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
        <nonDerivativeHolding>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>4407</value>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>I</value>
                </directOrIndirectOwnership>
                <natureOfOwnership>
                    <value>By the VNB Trust IRA fbo Mark T. Giles</value>
                </natureOfOwnership>
            </ownershipNature>
        </nonDerivativeHolding>
    </nonDerivativeTable>

    <footnotes>
        <footnote id="F1">In connection with certain personal investment, financial and tax planning strategies, (i) on December 27, 2016, PANDA Holdings, LLC (&quot;PANDA&quot;), of which the Reporting Person is the managing member, assigned 290,580 shares of the Issuer's common stock to an affiliate of PANDA for aggregate consideration of $1.00 and (ii) on December 28, 2016, such affiliate sold to MTG Investment Holdings, LLC (&quot;MTGIH&quot;), of which the Reporting Person is the sole member, 290,580 shares of the Issuer's common stock for aggregate consideration of $1.00.</footnote>
        <footnote id="F2">The Reporting Person is the managing member of PANDA and may be deemed to be the beneficial owner of such securities.  Mr. Giles disclaims beneficial ownership of such securities except to the extent of his pecuniary interest therein.</footnote>
        <footnote id="F3">The Reporting Person is the sole member of MTGIH and may be deemed to be the beneficial owner of such securities.  Mr. Giles disclaims beneficial ownership of such securities except to the extent of his pecuniary interest therein.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>/s/ Ben Shealy, attorney-in-fact for Mark T. Giles</signatureName>
        <signatureDate>2016-12-28</signatureDate>
    </ownerSignature>
</ownershipDocument>
