Aim To establish an efficient and stable method for isolation,culture and directional differentiation of endothelial progenitor cells from mouse bone marrow. Methods Mononuclear cells were isolated from mouse bone marrow by density gradient centrifugation and differential adhesion method.The remaining cells were cultured and differentiated to endothelial progenitor cells in EBM-2.The expressions of specific antigens(CD34,CD133,Flk-1 and CD31) on cell surface were analyzed by flow cytometer. Results Cells were obtained from mouse bone marrow by density gradient centrifugation and differential adhesion method formed clusters at day 4.At day 12,positive ratios of CD34+,CD133+,Flk-1+ and CD31+ were 65%±4%,48%±3%,37%±3% and 51%±4%,respectively. Conclusion An efficient,stable and replicable method for isolation and culture of endothelial progenitor cells from mouse bone marrow has been established.