Accelerating proliferation of primary keratinocytes benefits skin autografts for severely burned patients. Wnt signal, a conserved pathway controlling cell cycle and morphogenesis in embryo, also involves in cell proliferation and tumorigenesis in adult tissues. Here the effects of Wnt signal on the growth of human interfollicular keratinocytes were investigated. We demonstrated that recombinant Wnt3a significantly promoted the growth of primary keratinocytes at a low cell density. A well-characterized GSK-3b inhibitor, BIO, activated the Wnt signals and also enhanced the colony formation of keratinocytes dose dependently. Gene expression profile of the BIO-treated keratinocytes revealed the linkage of BIO with cell mitosis and indicated that epithelial cell adhesion molecule (EpCAM), a Wnt target gene, was significantly upregulated. Compared to the sorted EpCAM^- keratinocytes, the EpCAM⁺ cells showed a higher proliferation rate and efficacy of colony formation. Inhibiting the EpCAM expression by shRNA attenuated the proliferation effect of BIO and the growth advantage of the EpCAM⁺ keratinocytes. These evidences emphasize the positive roles of canonical Wnt and EpCAM on the regulation of cell growth and self-renewal of human keratinocytes.