Objective:To establish the cultural system of hMSCs in vitro,investigate the changes of Bmi-1 gene expression in different passages,and explore the effect of Bmi-1 in hMSCs replicative senescence. Methods:hMSCs were isolated from the femur bone marrow using adherence method and cultured in L-DMEM with 10% fetal bovine serum(FBS). The 4 th passage hMSCs were picked up for analyzing expressions of cell surface phenotype,and the 1 st,3 rd,6 th,9 th,12 th,15 th and 20 th passages were examined of Bmi-1 gene expression by RT-PCR. Results:hMSCs were successfully isolated from the femur bone marrow which were positive for CD13,CD105,CD33 and negative for CD34,CD38,CD117,CD45. The SA-β-gal staining showed that the senescent cells were more suffused than the vigorous cells. The Bmi-1 mRNA was detected in hMSCs, and the gene expression decreased related to the cell passage. Conclusion:hMSCs that can be successfully isolated from femur bone marrow are easy to proliferate. The replicative senescence of hMSCs has correlation with the expression level of Bmi-1 gene.