Objective：To investigate the effects of recombinant adenovirus-mediated co-infection of bone morphogenetic protein 9（BMP9） and vascular endothelial growth factor（VEGF） gene on osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）. Methods：C3H10T1/2 cells were infected by recombinant adenovirus-mediated of BMP9 and VEGF as follow five groups：Ad-BMP9 group，Ad-VEGF group，Ad-VEGF+Ad-BMP9 group，Ad-null group and control group. After infecting for 7 days，RT-PCR was performed to detect the mRNA expression level of BMP9 and VEGF，and real-time PCR was carried out to detect the mRNA ex－pression level of osteogenesis index osteocalcin（OC），osteoprotegerin（OPG），osteopontin（OPN）. Western blot was used to detect the protein expressions level of OPN. Alkaline phosphatase（ALP） activity was measured by quantitative and staining assay，and calcium deposition was determined by Alizarin Red S staining in each group. Results：BMP9 and VEGF co-expressed effectively in Ad-VEGF+Ad-BMP9 group. The mRNA level of OC，OPG，OPN and the protein level of OPN in Ad-BMP9 group and Ad-VEGF+Ad-BMP9 group were statistically higher than those of other groups（P<0.05），and were the highest in Ad-VEGF+Ad-BMP9 group（P<0.05）. The activity and staining assay of ALP，and the deposition of calcium in Ad-VEGF+Ad-BMP9 group were statistically higher than those in the other groups（P<0.05）. Conclusion：Combination of BMP9 and VEGF may promote osteogenic differentiation of C3H10T1/2 cells in vitro.