Simvastatin has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which simvastatin exerts its effects is still unclear. We previously reported that simvastatin promotes bone morphogenetic protein 2 (BMP-2) expression, induces osteoblastic differentiation, and inhibits adipocytic differentiation in mouse bone marrow stromal cells (BMSCs), and that this occurs, at least in part, via a BMP-2-dependent pathway. The aim of this study was to investigate further the mechanisms by which simvastatin stimulates osteogenesis in mouse BMSCs. To determine whether simvastatin-mediated osteogenesis was dependent on BMP-2, mouse BMSCs were treated with nonimmune normal mouse IgG or BMP-2 neutralizing antibodies combined with different concentrations of simvastatin. Surprisingly, the stimulatory effect of simvastatin on alkaline phosphatase (ALP) activity was not completely blocked by neutralizing BMP-2 monoclonal antibody treatment. Interestingly, we found that estrogen receptor-alpha (ER-alpha) protein levels increased after mouse BMSCs were treated with simvastatin for 72 h in a concentration-dependent manner. Moreover, the stimulatory effect of simvastatin on ALP activity in BMSCs was blocked by the estrogen receptor agonist ICI 182,780, and cotreatment with 17-beta-estradiol and simvastatin increased ALP activities by two-to threefold in the BMSCs compared with treatment with simvastatin alone. These results suggest that simvastatin-induced in vitro osteogenesis in mouse BMSCs is mediated, at least in part, by induction of ER-alpha and not by BMP-2 alone. These results provide new insight into the mechanisms of simvastatin-induced bone formation in BMSCs.