Skin tissue homeostasis is maintained by the balanced proliferation and differentiation of certain types of proliferating cells such as epidermal stem cells (EpSCs). The proliferation and differentiation of EpSCs are complex processes which are not well understood. This study aimed to find the internal relationship between the Nanog pathway and the Wnt/β-catenin pathway in the proliferation and differentiation process of EpSCs. In brief, EpSCs were isolated from rat epidermis and cultured. The MTT assay, western blotting, polymerase chain reaction (PCR) and immunocytochemistry were performed during the proliferation and differentiation process of EpSCs. Our results showed that 10⁻⁷ M neuropeptide substance P could effectively stimulate proliferation of EpSCs and that a possible link exists between the Nanog pathway and the Wnt/β-catenin pathway.