Hexosamine biosynthetic pathway (HBP) accounts for some cardiovascular adverse effects of hyperglycemia. We investigated whether HBP inhibitor azaserine protects against hyperglycemia-induced endothelial damage dependently of HBP. Human endothelial cells isolated from umbilical veins were exposed either to high (30.5 mmol/L) or low concentration of glucose (5.5 mmol/L) for 4 days followed by stimulation with TNF- (1 ng/ml, 24 hours). Blockade of the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT), inhibited HBP flux, oxidative stress (generation of superoxide and peroxynitrite) under the hyperglycemic condition, prevented the synergistic stimulation of VCAM-1 and ICAM-1 expression by hyperglycemia and TNF-. In the cells cultured under low glucose condition, when no increased HBP flux occurred, azaserine enhanced the manganese-superoxide dismutase (MnSOD) protein level, also inhibited the oxidative stress and the expression of VCAM-1 and ICAM-1 in response to TNF-. Moreover, the polyphenol resveratrol inhibited the oxidative stress and adhesion molecule expression, did not decrease the HBP flux under hyperglycemia condition. In addition, in isolated rat aortas exposed to hyperglycemic buffer for 5 hours when no significant HBP flux occurred, azaserine up-regulated MnSOD protein level and prevented decreased endothelium-dependent relaxations to acetylcholine. In conclusion, hyperglycemia independently increases oxidative stress and HBP flux, amplifies endothelial inflammation and impairs endothelial function mainly through oxidative stress and not the HBP pathway. Azaserine protects against hyperglycemic endothelial damage through its anti-oxidant effect independently of inhibiting HBP pathway.