Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.