To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail‐suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS^+/+ and iNOS^+/− mice responded to subsequent 14‐day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS^−/− mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS^−/− mice by treatment with an NO donor nitroglycerine (NG). Conversely, the responses in iNOS^+/+ mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone marrow cell cultures, mineralized nodules derived from iNOS^−/− mice after reloading were significantly reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension.