Objectives To investigate the effect of the combination of LMP-1 and HIF-1 alpha delivered by adipose-derived stem cells (ADSCs) on osteogenesis in vitro and in vivo. Results Cells expressing both LMP-1 and HIF-1 alpha genes had elevated mRNA expression of BMP-2, RunX2, alkaline phosphatase, osteocalcin, collagen I and alkaline phosphatase activity compared to cells from other groups. Furthermore, mineralization at day 14 in the cells expressing both LMP-1 and HIF-1 alpha was significantly higher than in all the other groups. In vivo, H&E staining and immunohistochemical analysis of the cell-scaffolds also showed more ectopic bone formation at 4 weeks compared to other groups. More new vessel formation was apparent in the pLVX-rHIF-1 alpha and pLVX-rLMP-1-rHIF-1 alpha groups. Conclusion LMP-1 and HIF-1 alpha gene delivery synergistically enhanced the osteo-differentiation of ADSCs in vitro and promoted osteogenesis in vivo compared with LMP-1 alone or HIF-1 alpha alone.