Introduction: Bone morphogenetic proteins (BMPs) are expected to be applied to bone regeneration therapy. BMP9 is one of the most potent among the 14 BMP members in inducing osteogenic differentiation, but its mechanism of action has not been fully elucidated. Our laboratory has recently revealed that BMP9 increases the protein expression of Hes1 in osteoblasts. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known effector of Notch signaling. In this study, I investigate the molecular mechanisms of Hes1 induction by BMP9 in osteoblasts. Methods: A mouse osteoblast cell line, MC3T3-E1, was stimulated with recombinant mouse BMP9. Hes1 mRNA and protein expression levels were analyzed by real time PCR and Western blotting. Next, MC3T3-E1 cells were stimulated with BMP9 in the presence of various signal inhibitors and their effects on BMP9-induced Hes1 expression were examined. Furthermore, Hes1 expression was knocked-down by transfecting siRNA against Hes1 into osteoblasts before BMP9 stimulation. Results: Stimulation of osteoblasts with BMP9 induced periodic increases of mRNA and protein expression of Hes1. Among the signal inhibitors, pretreatment with SMAD inhibitor signifi-cantly inhibited the Hes1 expression in BMP9-stimulated osteoblasts. Notably, the expression of osteogenic differ-entiation markers was highly increased by Hes1 siRNA in BMP9-stimulated osteoblasts. Conclusion: BMP9 induces the expression of Hes1 in osteoblasts via the SMAD pathway which is known as the canonical signal of BMPs. It was also found that the BMP9-induced expression of Hes1 plays an important functional role in the intracellular signaling events in osteoblasts stimulated by BMP9.