Serum response factor (SRF) is a transcription factor that transactivates actin‐associated genes and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in OL differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the micro‐RNAs (miRNAs) miR‐9 and the miR‐200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG‐1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Overexpression of miR‐9 and miR‐200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co‐expression of miR‐9 and miR‐200 attenuated activity of a luciferase reporter assay containing the Srf 3′ untranslated region. Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation. © 2012 Wiley Periodicals, Inc.