Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-alpha5beta1-mediated actin reorganization. Ectopic expression of integrin alpha5 in integrin-alpha5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin alpha5 (hereafter referred to as alpha5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin alpha5 engagement with fibronectin phosphorylated Ser643 of PKCdelta, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCdelta inactivation, dominant-negative PKCdelta adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of alpha5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCdelta expression sensitized alpha5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin alpha5, but not alpha5/1, mediates PKCdelta phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.