Objective To evaluate the effectiveness of adenovirus mediated human bone morphogenetic protein-2 gene transferred mesenchymal stem cells in the repair of segmental bone defect of long bone. Methods Unilateral rat femoral segmental defect model was used in this study.Thirty-six male Wistar rats were divided randomly into three groups:(1)Adv-hBMP-2 transduced MSC-collagen mix group; (2)Adv-βgal transduced MSC-collagen mix group; (3) untransduced MSC-collagen mix group. Contracted MSC-collagen mix was implanted in the defect site.The 4×106 cells were transplanted in each rat. Serial radiographs of rat femurs were examined at two, four, six and eight weeks postsurgery.Harvested specimens eight weeks postoperatively underwent histological and biomechanical analysis. Results The defects in group I (MSC/Adv-hBMP-2) appeared bone formation radiographically at two weeks postoperatively and united by eight weeks postoperatively. Histological analysis revealed that the defects in group I were filled with coarsd trabecular bone at eight weeks postoperatively. In those of the two control groups demonstrated little or no bone formation. Biomechanical test demonstrated that bending strength of femurs of the study group was 85%±8% to the opposite unoperated side at eight weeks postoperatively. Conclusion Adenovirus mediated bone morphogenetic protein-2 gene transferred syngeneic mesenchymal stem cells can repair segmental defects of long bones.