Objective In vitro isolation and culture of epidermal stem cells (ESCs) and transplantation of ESCs into skin injury model mice to explore their effect on skin wound healing. Methods Circumcised skin tissue was taken, and ESCs were separated and cultured by adherent type IV collagen; 3T3 cells were used as the feeder layer to sort ESCs. A mouse skin injury model was established by dorsal trauma method , and mice were randomly divided into a normal control group, model group, and spot injection cell group. The wound healing rates were recorded on days 0, 3, 7, 11, and 15 of treatment. On the 16th day of treatment, we determined the contents of SOD, GSH, GSH-Px, and MDA in the tissues. ELISA was used to determine IL-10, TGF-β, and TNF-α levels in the serum. HE staining was used to observe histopathological changes in the skin wounds. Results On the 7th and 11th days of treatment, the wound area in the spot injection cell group group was smaller than that in the model group (P<0. 05), and the spot injection cell group’s wounds were almost healed on the 15th day (98. 08% ± 1. 77%). Compared with the model group, the spot injection cell group’s contents of SOD, GSH, GSH-Px were significantly increased (P<0. 01), and MDA was significantly reduced (P<0. 01); the ELISA result showed that the serum contents of IL-10 and TGF-β were increased (P<0. 05) and TNF-α content had declined in the spot injection cell group (P<0. 05). The result of HE staining showed that, compared with model group, the spot injection cell group had reduced infiltration of inflammatory cells and more hair follicles and collagen. Conclusions ESCs can accelerate wound epithelialization and effectively promote wound healing.