Abstract： Objective To establish the two-dimensional gel electrophoresis (2-DE) map of keratinocytes and epidermal stem cells, to analyze the differentially expressed proteins between two type cells and provide clues for regulating the proliferation and differentiation of epidermal stem cells in vitro. Methods The single epidermal cell suspension was obtained by enzyme digestion and the epidermal stem cells were harvested based on collagen type Ⅳ rapidly adhesiveness. The protein expression maps of the two types of cells were obtained using 2-DE, and the differential expressed protein spots were analyzed by linage master 2D elite 5.0 software. Results There were good reproducibility and comparability between two cell lines from the 2-DE protein expression maps. Average protein spots were 982 ±18 in keratinocytes and 930 ± 15 in epidermal stem cells. The matched spots were 850 ± 13 and 798 ±11 in these two types of cells, and the average matching rates were 86.56% and 85.81%. There were 886 ± 8 matching protein spots between two types of cells, and the average matching rate was 76.98%. Eleven differential protein spots were identified between two types of cells. Eight protein spots were expressed only or higher in the epidermal stem cells and three protein spots were expressed only or higher in the keratinocytes. Conclusion The proteome maps of keratinocytes and epidermal stem cells with high resolution and reproducibility can be obtained by the technique of 2-DE and some differences exist in protein expression between these two types of cells.