Aim To explore the protective effect of Vitamin C on the human umbilical vein endothelial cell (hUVECs) injured by lipid peroxidation and its probable mechanism. Methods Confluent hUVECs were incubated with Vitamin C for 24 h and consequently exposed to lipid peroxides induced by high concentration of Copper ions. Lactate dihydrogenase (LDH) activity was measured in cell medium and lysate. Endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production induced by acetylcholine were assessed by endothelial nitric oxide synthase and nitric oxide kits. Results Lipid peroxides enhanced markedly LDH activity and decreased nitric oxide bioavailability via the inhibited eNOS activity (p<0.01). These effects were attenuated by administration of different dose Vitamin C (100 μmol/L, 300 μmol/L); the correlation between eNOS activity and NO production was significant (r=0.9844, p<0.01; r=0.8802, p<0.05, respectively). Conclusions Lipid peroxides could injury hUVECs and inhibit eNOS activity directly. Vitamin C might play a protective role in hUVECs injured by lipid peroxidation.