Reduced mechanical stress leads to bone loss, as evidenced by disuse osteoporosis in bedridden patients and astronauts. Osteocytes have been identified as major cells responsible for mechanotransduction; however, the mechanism underlying the response of bone to mechanical unloading remains poorly understood. In this study, we found that mechanical unloading of wildtype mice caused decrease of Wnt/β-catenin signaling activity accompanied by upregulation of Sost. To further analyze the causal relationship among these events, Sost gene targeting mice were generated. We showed that sclerostin selectively inhibited Wnt/β-catenin in vivo, and sclerostin suppressed the activity of osteoblast and viability of osteoblasts and osteocytes. Interestingly, Sost-/- mice were resistant to mechanical unloading-induced bone loss. Reduction in bone formation in response to unloading was also abrogated in the mutant mice. Moreover, in contrast to wildtype mice, Wnt/β-catenin signaling was not altered by unloading in Sost-/- mice. Those data implied that sclerostin played an essential role in mediating bone response to mechanical unloading, likely through Wnt/β-catenin signaling. Our findings also indicated sclerostin is a promising target for preventing disuse osteoporosis. © 2009 American Society for Bone and Mineral Research.