Objective To optimize the culture conditions of the human bone marrow mesenchymal stem cells(hMSCs). Methods The influence of the primary culture method, planting density, the time of the first medium changing , culture medium and serum concentration on growth of the hMSCs were analyzed. Results When the other conditions were the same, the density gradient isolation was better than whole-marrow isolation;2.5×105 cells/cm2 was the best planting density;the best first medium changing was the fifth day at primary culture, DMEM medium was better than α-MEM, serum C was the best of four serums compared, 10% was the suitable serum concentration. The hMSCs under the optimal conditions could expand over 15 passages, remaining their normal modality and differentiation potentials. Conclusion The optimal culture condition of the hMSCs is established and it is a new investigation on application of hMSCs to tissue engineering.