The epidermis, the outermost layer of the skin, is the first barrier that comes into contact with the external environment. It plays an important role in resisting the invasion of harmful substances and microbial infections. The skin changes with age and external environmental factors. This study aimed to investigate epidermal stem cells during the process of aging. This study enrolled 9 volunteers with benign pigmented nevus for clinical dermatologic surgery. The phenotypes associated with skin aging changes such as skin wrinkles and elasticity of the unexposed/healthy parts near benign pigmented skin were measured, and epidermal stem cells from this region were isolated for transcriptome sequencing. The results showed that epidermal stem cells could be obtained by magnetic activated cell sorting (MACS) with high purity. Results of the transcriptome sequencing revealed that aquaporin (AQP)5 significantly decreased in the epidermal stem cells with age, and further functional experiments revealed that AQP5 could promote the proliferation and dedifferentiation of HaCaT, but did not influence cell apoptosis. In summary, AQP5 regulated the proliferation and differentiation of epidermal stem cells in skin aging, and it may play an important role in the balance of proliferation and differentiation. However, further studies are needed to determine the mechanism by which AQP5 regulates the proliferation and differentiation of epidermal skin cells in aging.