Reports of the bone‐protective effects of resveratrol, a naturally occurring phytoestrogen and agonist for the longevity gene SIRT1, have highlighted this compound as a candidate for therapy of osteoporosis. Moreover, SIRT1 antagonism enhances adipogenesis. There has been speculation that resveratrol can promote osteogenesis through SIRT1, but the mechanism remains unclear. In this study we investigated the molecular mechanism of how resveratrol can modulate the lineage commitment of human mesenchymal stem cells to osteogenesis other than adipogenesis. We found that resveratrol promoted spontaneous osteogenesis but prevented adipogenesis in human embryonic stem cell–derived mesenchymal progenitors. Resveratrol upregulated the expression of osteo‐lineage genes RUNX2 and osteocalcin while suppressing adipo‐lineage genes PPARγ2 and LEPTIN in adipogenic medium. Furthermore, we found that the osteogenic effect of resveratrol was mediated mainly through SIRT1/FOXO3A with a smaller contribution from the estrogenic pathway. Resveratrol activated SIRT1 activity and enhanced FOXO3A protein expression, a known target of SIRT1, in an independent manner. As a result, resveratrol increased the amount of the SIRT1‐FOXO3A complex and enhanced FOXO3A‐dependent transcriptional activity. Ectopic overexpression or silencing of SIRT1/FOXO3A expression regulated RUNX2 promoter activity, suggesting an important role for SIRT1‐FOXO3A complex in regulating resveratrol‐induced RUNX2 gene transcription. Further mutational RUNX2 promoter analysis and chromatin immunoprecipitation assay revealed that resveratrol‐induced SIRT1‐FOXO3A complex bound to a distal FOXO response element (−1269/−1263), an action that transactivated RUNX2 promoter activity in vivo. Taken together, our results describe a novel mechanism of resveratrol in promoting osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis. © 2011 American Society for Bone and Mineral Research