Runt-related transcription factor 2 (Runx2) is a key transcription factor for osteogenic gene expression and osteoblast differentiation. In order to maintain bone homeostasis, the transcriptional activity of Runx2 is tightly modulated by many intra- and extra-cellular factors. Here, we reveal the mechanism by which Hairy Enhancer of Split1 (Hes1) regulates the transcriptional activity of Runx2, and elucidate the potential role of Hes1 during osteoblast differentiation. Coexpression of Hes1 with Runx2 promoted an increase in Runx2 protein levels by increasing the half-life of Runx2; Hes1 thereby augmented the formation of a Runx2-DNA complex at Runx2 target sites. During osteoblast differentiation, the retroviral overexpression of Hesl accelerated osteogenesis and stimulated the expression of osteogenic marker genes, including osteopontin and type I collagen. Taken together, these results suggest that Hes1 augments the protein level and transcriptional activity of Runx2, resulting in the stimulation of osteoblast differentiation. (C) 2007 Published by Elsevier Inc.