Protein kinase C (PKC) α has been implicated in β1 integrin‐mediated cell migration. Stable expression of PKCα is shown here to enhance wound closure. This PKC‐driven migratory response directly correlates with increased C‐terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3‐kinase blockade. Upon phorbol 12, 13‐dibutyrate stimulation, green fluorescent protein–PKCα and β1 integrins co‐sediment with ERM proteins in low‐density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCα is shown to form a molecular complex with ezrin, and the PKC‐co‐precipitated endogenous ERM is hyperphosphorylated at the C‐terminal threonine residue, ie activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C‐terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCα‐induced cell migration. We provide the first evidence that PKCα or a PKCα‐associated serine/threonine kinase can phosphorylate the ERM C‐terminal threonine residue within a kinase–ezrin molecular complex in vivo.