The aim of the present study was to test the osteogenic effects of BMP‐2 (bone morphogenetic protein‐2) gene transfer in BMSCs (bone‐marrow stromal cells) and rabbit calvarial bone defects. The pBMP‐2‐cDNA3.1 plasmid was constructed by subcloning hBMP‐2 (human BMP‐2) cDNA into the plasmid pcDNA3.1. BMSCs were transfected with a pBMP‐2‐cDNA3.1–Lipofect‐amine™ complex. Transfected cells were observed for localization of the BMP‐2 coding plasmid. Also, the level of BMP‐2 in the culture medium of transfected cells was measured. The culture medium was collected and we tested whether this medium could induce non‐transfected BMSCs to express ALP (alkaline phosphatase) and osteocalcin. The pBMP‐2‐cDNA3.1 complexes were incorporated into the collagen scaffold and the plasmid‐loaded collagen scaffolds were then grafted into rabbit calvarial defects. After 2 weeks, granulation tissue at the grafted site was obtained and mRNA of BMP‐2 was examined via RT (reverse transcriptase)–PCR. After 4 and 8 weeks, the animals were killed and the calvarial tissue was excised. After specimen preparation, optical microscopical examination was performed to evaluate bone formation. The results show that transfected cells were able to incorporate the BMP‐2 gene into their nuclei. Also, the level of expressed and secreted BMP‐2 was significantly higher in transfected cells than in untransfected cells (P<0.01). The retrieved culture medium could induce the expression of ALP and osteocalcin in non‐transfected BMSCs. hBMP‐2 mRNA was detected at the granulation tissue of experimental animals, but not in control animals after 2 weeks. At both 4 and 8 weeks, experimental groups showed significantly more newly formed bone area than the control group (P<0.01). Therefore pBMP‐2‐cDNA3.1 gene delivery could induce BMSCs into osteoblastic phenotype cells and enhance bone regeneration in rabbit calvarial bone defects.