The high-level production of nitric oxide (NO) induced by inflammatory cytokines has been shown to play a key role in the pathogenesis of inflammation-mediated osteoporosis. In the present work, we observed that 1 mM of the NO donor sodium nitroprusside (SNP) induced an increase of the cytosolic calcium concentration ([Ca2+]c) in osteoblasts, which was completely abolished by applying an extracellular Ca2+-free buffer. Further experiments showed that the SNP-induced [Ca2+]c increase was specifically blocked by potent antagonists of the transient receptor potential vanilloid subtype 1 (TRPV1) channel: capsazepine, ruthenium red, and La3+ in Ca2+-containing buffer. However, nifedipine, an L-type voltage sensitive Ca2+-channel blocker, failed to suppress the [Ca2+]c elevation caused by SNP. Additionally, 1 mM SNP induced osteoblast apoptosis, which was largely inhibited by the blockers of TRPV1, capsazepine and ruthenium red. Interestingly, our data showed that the SNP-induced [Ca2+]c increase was significantly inhibited by N-ethylmaleimide, the blocker of S-nitrosylation modification, instead of inhibitors of the NO–cGMP–PKG pathway. Taken together, our data clearly demonstrated that the NO donor SNP resulted in apoptosis associated with TRPV1 channel-mediated Ca2+ entry via S-nitrosylation in osteoblasts.