Abstract: Objective To construct tissue-engineered skin via in vitro inoculation of epidermal stem cells （ESCs） onto de-epidermized dermis. Methods Skin tissue was obtained from the foreskin of a healthy 6-year-old child, and keratinocytes were isolated by two-step trypsinization method followed by the collection of ESCs via rapid adhesion by collagen Ⅳ. The ESCs were identified by morphological observation and immunohistochemical staining with K19 and integrin β1. To construct tissue-engineered skin, selected ESCs were seeded onto the surface of de-epidermized dermis followed by an one-week culture immersed in the medium and a subsequent 4-week culture at the air-medium interface. The tissue-engineered skin was evaluated with haematoxylin & eosin （HE） staining as well as keratin immunohistochemistry. Results Microscopically, cultured ESCs showed a paving stone-like appearance and grew into colonies. Immunohistochemistry revealed that the ESCs were positive for integrin-β1 and keratin 19. After 5 weeks of culture, 3 ~ 6 layers of epidermal cell were observed on the dermis with the formation of stratum corneum. Keratin protein was observed in the artificial epidermal skin. Conclusion Tissue-engineered skin is successfully constructed with epidermal stem cells and de-epidermized dermis in vitro .