BACKGROUND: Double gene transfection using bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165) for bone marrow stromal stem cells (BMSCs) to induce osteogenesis provides experimental basis for the study on tissue engineering bone. OBJECTIVE: To investigate the ability of BMP2 and VEGF165 double gene modified rat BMSCs to induce osteogenesis. METHODS: BMSCs were isolated from the femur and tibia of four 4-week-old Sprague-Dawley rats by whole bone marrow adherent culture. Passage 3 BMSCs were randomized into five groups: non-transfection group, empty plasmid group, BMP2 transfection group, VEGF165 transfection group, BMP2 and BMP2/VEGF165 transfection group (co-transfection group). Then, western blot assay was used to detect expression of BMP2 and VEGF165 at 48 hours after transfection, and the activity of alkaline phosphatase was detected in each group at 7 days after transfection. RESULTS AND CONCLUSION: Highly expressed BMP2 in BMP2 and co-transfection groups and highly expressed VEGF165 in VEGF165 and co-transfection groups were found after transfection. The expression of BMP2 or VEGF165 in the co-transfection group was significantly higher than that in the BMP2 or VEGF165 transfection group after transfection, respectively (P < 0.05). Western blot results showed that the activity of alkaline phosphatase was ranked as: co-transfection group > BMP2 transfection group > VEGF165 transfection group > empty plasmid group and non-transfection group. There was a significant difference in the activity of alkaline phosphatase between co-transfection group and any of single gene transfection groups (P < 0.05). To conclude, BMP2/VEGF165 co-transfection promotes the ectopic osteogenesis of BMSCs.