Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease. The S-nitrosylation of proteins is involved in the regulation of cardiovascular functions. However, whether homocysteine (Hcy) impairs vascular functions through the inhibition of protein S-nitrosylation in the endothelium remains to be determined. The experiments were performed in human umbilical vein endothelial cells (HUVECs). Male Sprague‑Dawley rats, with or without administration of L-methionine, were used for the in vivo validation of findings. S-nitrosylation was analyzed using immunofluorescence for nitrosocysteine, and further confirmed by the biotin switch method. The levels of reactive oxygen species (ROS) were detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. The levels of nitric oxide (ΝΟ) were determined by the nitrate reduction method. Protein expression was analysed by western blot analysis. The activity of nuclear factor κB (NF-κB) was evaluated by an electrophoretic mobility shift assay (EMSA). The levels of plasma Hcy were measured by ELISA. The results showed that Hcy significantly reduced the levels of protein S-nitrosylation in HUVECs and endothelial S-nitrosylation of aorta. This reduction of protein S-nitrosylation was accompanied by increasing ROS, decreasing phosphorylation levels of Akt and endothelial nitric oxide synthase (eNOS), and reduced levels of nitric oxide in HUVECs. In addition, it was found that Hcy increased the protein expression of vascular cell adhesion molecule-1 by attenuating the cytoplasm S-nitrosylation of NF-κB (p65). These data suggested that Hcy impairs endothelial functions by inhibiting endothelial protein S-nitrosylation.