The aim of this study was to investigate the expression of bone morphogenetic protein-2 (BMP-2) in bone marrow stromal stem cells (BMSCs) and the in vivo and in vitro osteogenic activity of BMSCs transfected with the adenovirus plasmid, Ad-GFP-hBMP-2. The Ad-GFP-hBMP-2 plasmid was packaged and transfected into rabbit BMSCs to determine the transfection rate. The alkaline phosphatase (ALP) activities of Ad-GFP-hBMP-2-transfected BMSCs (experimental group) and untransfected BMSCs (control group) were detected. In situ hybridization of type I collagen and Western blot were used to determine the BMP-2 gene and protein expressions. The transfected and untransfected BMSCs were respectively inoculated into nude mice to observe in vivo osteogenesis. The decalcified bovine cancellous bone scaffold was respectively combined with transfected and untransfected BMSCs and implanted into ulnar defects in rabbits to repair the bone. The adenovirus titer was 1.2x10(10) pfu/mL. Green fluorescent protein expression appeared 48 h after transfection with the adenovirus plasmid, and the transfection rate was 71.1%. The ALP activity was higher in the experimental group than the control group at each time point after transfection. The gene and protein expressions of BMP-2 were higher in the experimental group than the control group. The positive rates of in vivo osteogenesis in the experimental and control groups were 90% and 40%, respectively. The bone defect repair effects differed markedly between the two groups. The BMP-2 gene can be highly expressed in BMSCs to successfully induce osteogenic differentiation. BMSCs can be used as seed cells for bone tissue engineering.