The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCalpha, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCalpha into the nucleus, where PKCalpha exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCalpha in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.