Objective Isolation and culture of Kunming mouse embryonic fibroblasts (MEFs) for growth feature in vitro are used to culture the embryonic stem cell. Methods Kunming MEFs were isolated and cultured by digestion.MTT assay was used to plot the growth curve of each generation of cells.The cytoskeleton of each generation of cells was analyzed by immunofluorescence.The MEFs were dealt with different concentration of Mitomycin C(10 μg/mL

20 μg/mL and 30 μg/mL) for 1 hour

2 hours and 3 hours.The optimum condition of feeder layer preparation was screened by MTT assay.The feeder layer was prepared for the culture of mouse embryonic stem cells. Results The proliferation ability of generation 3 to 5 of MEFs was good.The cytoskeleton immunofluorescence staining showed that the generation 1 to 3 of cell microtubules and microfilaments arranged in neat rows，the generation 5 to 7 of cells microtubules and microfilaments were disorganized.The feeder cells dealt with 10 μg/mL Mitomycin C for 2 hours was better for the culture of mouse embryonic stem cell.Conclusion The generation 3 to 5 of MEFs can be used for the culture of mouse embryonic stem cells.