Hyperhomosysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice, but not in the p53-deficient cells. The NO donor S-nitroso-N-acetlylpenicillamine, adenoviral iNOS vector (AdiNOS) transfection, and antioxidants (-tocopherol and superoxide dismutase plus catalase), but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by siRNA-mediated suppression of Noxa expression, indicating that Noxa upregulation plays an important role in HCy-induced endothelial cell apoptosis. AdiNOS transfection increased the formation of S-nitrosoHCy (S-NOHCy), which was inhibited by the NOS inhibitor N-monomethyl-L-arginine. Moreover, S-NOHCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.