Introduction: Bone morphogenetic proteins (BMPs) are known to be involved in the processes of bone healing and regeneration. Latest reports showed that BMP9 possess the strongest osteoinductive potential among BMPs. Hif-1αis a well-established hypoxia-responsive protein inducing angiogenic, and plays important roles in skeletal devel-opment. Our study aimed to investigate the functional roles of Hif-1α in the signaling pathway of BMP9-induced osteoblast differentiation. Methods: MC3T3-E1 cells (a mouse osteoblast cell line) were stimulated with recombi-nant BMP9. We then stimulated MC3T3-E1 cells with BMP9 under normoxic or hypoxic (2% O2) condition with or without Chrysin (PHD activator) or NSC697923 (Ubc13 inhibitor). Hif-1α gene knockdown was performed by transfecting MC3T3-E1 cells with Hif-1α-specific siRNA. Protein and mRNA expression levels were analyzed by Western blotting and real-time PCR, respectively. We also analyze the mineralization process of BMP9-induced os-teoblasts in the absence or presence of Hif-1α inhibitor with Alizarin red staining. Results: Hif-1α protein expression was significantly induced by both BMP9 in MC3T3-E1 cells within 2h. Combination of BMP9 and hypoxia resulted in a synergistic increase of Hif-1α protein level. Although Chrysin inhibited both BMP9- and hypoxia-induced Hif-1αprotein expressions, NSC697923 failed to inhibit BMP9-induced Hif-1α protein expression. When Hif-1α expression was knocked down by siRNA, the mRNA expression level of ALP was affected in BMP9-stimulated MC3T3-E1 cells but not Runx2 and Osterix. Mineralization process of osteoblasts was inhibited in the present of Hif-1α inhibitor. Conclusions: We have revealed that the protein expression of Hif-1α in osteoblasts is rapidly induced by osteogenic BMPs under normoxic condition. Hif-1α expression is essential for the induction of ALP, but not that of osteogenic transcription factor, Runx2 and Osterix in BMP9-stimulated osteoblasts. Our findings indicate for the first time that Hif-1α expression is important in BMP9-mediated osteoblast differentiation not by means of osteogenic transcription factor, Runx2 or Osterix.