Actin remodeling plays a crucial role in insulin-induced translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the plasma membrane and subsequent glucose transport. Protein kinase C (PKC) zeta has been implicated in this translocation process, although the exact mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin cytoskeleton and translocation of GLUT4 in CHO-K1 cells expressing myc-tagged GLUT4. Insulin stimulated the phosphorylation of PKCzeta at Thr410 with no apparent effect on its protein expression. Moreover, insulin promoted colocalization of PKCzeta and actin that could be abolished by Latrunculin B. The overexpression of PKCzeta mimicked the insulin-induced change in actin cytoskeleton and translocation of GLUT4. These effects were also completely abrogated by Latrunculin B treatment. Using cell-permeable pseudosubstrate (PS) inhibitor of PKCzeta, the response to insulin could be alleviated. Our results strongly suggest that PKCzeta mediates the stimulatory effect of insulin on GLUT4 translocation through its interaction with actin cytoskeleton.