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Simvastatin, an HMG-CoA reductase inhibitor, is known to promote osteogenic differentiation. However, the mechanism underlying simvastatin-induced osteogenesis is not well understood. In this study, we hypothesize that the estrogen receptor (ER) mediates simvastatin-induced osteogenic differentiation. ER antagonists and siRNA were used to determine the involvement of the ER in simvastatin-induced osteogenesis in mouse bone marrow mesenchymal stem cells (D1 cells). Osteogenesis was evaluated by mRNA expression, protein level/activity of osteogenic markers, and mineralization. The estrogen response element (ERE) promoter activity and the ER–simvastatin binding affinity were examined. Our results showed that the simvastatin-induced osteogenic effects were decreased by treatment with ERα antagonists and ERα siRNA but not by an antagonist specific for the G protein-coupled estrogen receptor (GPER-1). The simvastatin-induced osteogenic effects were further increased by E2 treatment and were reversed by ERα antagonists or siRNA treatment. Luciferase reporter gene assays demonstrated that simvastatin increase ERα-dependent transcriptional activity that was suppressed by ERα antagonists. Furthermore, the ERα–simvastatin binding assay showed that IC50 value of simvastatin is 7.85 μM and that of E2 is 32.8 nM, indicating that simvastatin is a weak ligand for ERα. These results suggest that simvastatin-stimulated osteogenesis is mediated by ERα but not GPER-1. Moreover, this is the first report to demonstrate that simvastatin acts as an ERα ligand and a co-activator to enhance ERα-dependent transcriptional activity and thus promotes osteogenesis. These results indicate that simvastatin-induced osteogenesis is mediated via an ERα-dependent pathway.