Objective To explore a feasible and efficient method for isolating rat fetal epidermal stem cells (rfESCs) , and to observe the differentiation potential of rfESCs in vivo. Methods The different development stages of SD rat fetal skin, ED14，ED16, ED18 and ED20, were collected and the structures were analyzed by HE staining. The rfESCs were isolated by rapidly adhering to rat tail collagen (RA-RTC) and were cultured in keratinocyteserum free medium(K-SFM). The expression of β1integrin and cytokeratin 19 in rfESCs were detected by immunhistochemical staining. Meanwhile, the cell cycle and proliferative capacity were also determined. To induce its differentiation, these cells were grafted onto porous gelatin sponge after labelled with a fluorescent dye (PKH26), and then transferred into rat subrenal capsule. Results Epidermis and dermis were incompletely differentiated until ED18. The rfESCs were effectively isolated from ED18 rat fetal skin by RARTC. The expressions of cytokeratin 19 and β1integrin in rfESCs were positive. Cell cycle analysis indicated a slow cell cycle. rfESCs were differentiated and exhibited epidermallike structure under renal capsule. Conclusion The early