AMP-activated protein kinase (AMPK) and Rho-kinase (ROK) are known to modulate the mevalonate pathway. Activation of AMPK suppresses HMG-CoA reductase. ROK acts on downstream of HMG-CoA reductase, and its inhibition exerts anti-atherosclerosis effects. However, whether or not these enzymes are involved in bone metabolism is unclear. The present study was undertaken to investigate the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide1--D-ribonucleoside (AICAR), and a ROK inhibitor, fasudil hydrochrolide, on the mineralization of osteoblastic MC3T3-E1 cells. Real-time PCR and mineralization stainings revealed that both AICAR and fasudil significantly stimulated endothelial nitric oxide synthase (eNOS), bone morphogenetic protein-2 (BMP-2), and osteocalcin mRNA expression as well as mineralization in the cells. Supplementation of either mevalonate or geranyl-geranyl pyrophosphate, the downstream molecules of HMG-CoA reductase, or co-incubation with either a NOS inhibitor, L-NAME, or a BMP-2 antagonist, noggin, significantly reversed these AICAR-induced reactions. Western blot analysis showed that AICAR activated Akt and extracellular signal-regulated kinase (ERK). ERK inhibitor significantly reversed the AICAR-induced increase in eNOS and BMP-2 mRNA expression. Measurement of ROK activities by ELISA revealed that both AICAR and fasudil significantly suppressed the phosphorylation of the myosin-binding subunit of myosin phosphate, a ROK substrate. These findings suggest that the AMPK activator as well as the ROK inhibitor are able to stimulate the mineralization of osteoblasts through modulating the mevalonate pathway. These agents could be candidate drugs that promote bone formation for the treatment of osteoporosis.