In vitro treatment with a pharmacological dose of simvastatin, a potent pro-drug of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, stimulates bone formation. In our study, simvastatin stimulated differentiation of osteoblasts remarkably in a dose-dependent manner, with minimal effect on proliferation. To identify the mediators of the anabolic effects of simvastatin on osteoblasts, we tried to identify and characterize simvastatin-induced proteins by using proteomic analysis. Calcyclin was significantly up-regulated by more than 10 times, and annexin I was also up-regulated by simvastatin. However, annexin III, vimentin, and tropomyosin were down-regulated. Up-regulated calcyclin mRNA by simvastatin was validated by reverse transcription in mouse calvarial cells. In confocal microscope analysis, green fluorescence protein-calcyclin fusion protein was ubiquitously observed in the of MC3T3-E1 cells transfected with green fluorescence protein-calcyclin cDNA containing plasmid and was quickly concentrated in the nucleus 20 min after simvastatin treatment. Overexpression of calcyclin cDNA stimulated both the proliferation and expression of alkaline phosphatase mRNA significantly, without exposure to simvastatin in MC3T3-E1 cells. However, both the rate of proliferation of the osteoblasts and the expression of alkaline phosphatase mRNA were suppressed significantly 1 day after treatment with the calcyclin-specific small interference RNA, and furthermore, simvastatin did not overcome this suppression in the small interference RNA-pretreated MC3T3-E1 cells. In conclusion, calcyclin is one of the candidate proteins that plays a role in osteoblastogenesis in response to simvastatin, although the precise functions of calcyclin in osteoblast remain to be verified.