Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the l ipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, weretransfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 ± 0.03, 0.90 ± 0.02, 0.64 ± 0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P lt; 0.05); there was no significant difference among groups C, D and E (P gt; 0.05). Conclusion The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation l iposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abil ities of BMSCs.