We reported previously that protein kinase C-α (PKC-α) negatively regulates Wnt/β-catenin signalling pathway. The current study explores the role of PKC-α in the regulation of proliferation of colon cancer cells, which contain aberrant up-regulation of intracellular β-catenin. In colon tissue and cells, an inverse correlation was observed between the expression levels of PKC-α and intracellular β-catenin. Activation of PKC-α inhibited β-catenin response transcription by down-regulation of intracellular β-catenin and induced phosphorylation of the N-terminal serine and threonine residues (Ser33/Ser37/Thr41) of β-catenin, marking it for proteasomal degradation, in colon cancer cells. Pharmacological inhibition or depletion of PKC-α-abrogated PKC-α-mediated β-catenin down-regulation and phosphorylation in colon cancer cells. Notably, the Ser45 residue of β-catenin was essential for PKC-α-induced β-catenin down-regulation in colon cancer cells. Moreover, PKC-α activation repressed the expression of cyclin D1 and c-myc, which are known β-catenin target genes, and thus inhibited the growth of colon cancer cells. These findings suggest that PKC-α negatively regulates colon cancer cell proliferation viaβ-catenin phosphorylation/down-regulation and may facilitate the development of new strategies to treatment of colon cancer.