Aim To evaluate the tyrosine nitration modification of specific proteins in vascular endothelial cells and its impact on mitochondria-mediated apoptosis. Methods Human umbilical vein endothelial cells were cultured in vitro and divided into three groups:control group (treatment with dimethyl sulfoxide), 3-morphansulam (SIN-1) group, and SIN-1＋Fe(III) 5,0,5,0-(tetraphenyl)porphyrin (FeTPP) group. After 24 h, the levels of hexokinase 1 (HK1) nitration modification, mitochondrial membrane potential, reactive oxygen species (ROS) production, and endothelial cell proliferation and apoptosis were assessed. A human umbilical vein endothelial cell line knockout of HK1 was constructed using gene editing technology, and its proliferation and apoptosis levels were detected. Results After treatment of human umbilical vein endothelial cells with peroxynitrite generator SIN-1, the level of HK1 protein nitration modification significantly increased (P＜0.01), reactive oxygen species production significantly increased, mitochondrial membrane potential significantly decreased, endothelial cell proliferation ability significantly decreased, and endothelial cell apoptosis level significantly increased (all P＜0.01). Peroxynitrite decomposition catalyst FeTPP could reverse the above effect (P＜0.01). In addition, HK1 gene knockout also exhibited similar antioxidant effects, with a significant decrease in endothelial cell proliferation ability and a significant increase in apoptosis levels (P＜0.01). Conclusion Peroxynitrite can induce an increase in the level of nitration modification of HK1 in vascular endothelial cells, which may be achieved by promoting the production of mitochondrial reactive oxygen species, thereby accelerating the process of endothelial cell apoptosis.