Delayed and failed bone union following fracture is a common clinical complication that requires treatment in orthopedics. Cell-based therapies and tissue-engineering approaches are potential therapeutic strategies for bone repair and fracture healing. However, the effect of adenovirus expressing bone morphogenetic protein-2 (Ad-BMP-2) on the osteogenic ability of human mesenchymal stem cells (hMSCs) has remained to be fully elucidated. Therefore, in the present study, hMSCs were transduced using Ad-BMP-2 to assess the effects of its application and to determine whether Ad-BMP-2 promotes the osteogenic differentiation of hMSCs. The purity of the hMSC cultures was assessed using flow cytometric analysis. In order to assess the osteogenic activity, alkaline phosphatase activity (ALP) was measured and to estimate the osteoblastic mineralization and calcification, von Kossa staining for phosphates was performed. Cells positive for Src homology 2 domain were determined to be hMSCs and the presence of CD34 was used to distinguish hematopoietic lineages. Following treatment, the Ad-BMP-2 and control group had significantly increased ALP levels (P<0.05). Compared to the blank group and the group transfected with adenoviral vector containing LacZ, the phosphate deposition in the Ad-BMP-2 group and the positive control group treated with dexamethasone was markedly increased. The results of the present study suggested that Ad-BMP-2 promotes osteogenic differentiation in hMSCs and may have a potential application in treating delayed union and nonunion following bone fracture.