Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.