To investigate the effect and mechanism of leptin (LEP) on the osteoblastic differentiation of hBMSCs in vitro.Whole bone marrow culture method was applied to culture hMBSCs and hBMSCs at passage 3 were divided into groups A, B, C, D, E and F, and when cell attachment was evident, 400, 200, 100 and 50 ng/mL LEP, 100 ng/mL BMP and common nutrient medium were added into each group, respectively. ALP staining and mineralized nodules staining were conducted at 7 and 21 days after culture, respectively. And inverted phase contrast microscope observation was performed. ALP activity and osteocalcin (OCN) level of hBMSCs in each group was detected at 7, 14 and 21 days after culture to select the best induced concentration of LEP on osteoblastic differentiation. For groups of B, E and F at 7 days after culture, RT-PCR was adopted to detect the expression of such osteogenesis-related genes as core-binding factor alpha 1 (Cbf alpha 1), ALP, Col I and OCN mRNA.At 7 days after induced culture, the ALP staining result showed that the endochylema in groups A, B, C, D and E were stained blue and the endochylema in the group F was slightly positive. At 21 days after induced culture, the mineralized nodules staining showed that cells in groups A, B, C, D and E were stained positively and cells in group F were negative. At 7, 14 and 21 days after culture, ALP and OCN activities in group B were less than that of group E (P < 0.05), but significant higher than that of groups A, C, D and F (P < 0.05), the optimal concentration of LEP was 200 ng/mL. At 7 days after culture, group F witnessed no expression of Cbf alpha 1, ALP, Col I and OCN mRNA, while groups B and E witnessed expressions of all those indexes, but the expressions in group B were less than those of group E.LEP can stimulate osteoblastic differentiation of hBMSCs in vitro, and the possible mechanism is its role of promoting the expression of osteoblastic related genes.