Endothelial cell dysfunction is considered a major cause of vascular complications in diabetes. In the present study, we investigated the protective effect of daidzein, a natural isoflavonoid, against high-glucose-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). Treatment with a high concentration of glucose (30 mM) induced oxidative stress in the endothelial cells, against which daidzein protected the cells as demonstrated by significantly increased cell viability. In addition, lipid peroxidation, intracellular reactive oxygen species (ROS) generation, and indirect nitric oxide levels induced by the high glucose treatment were significantly reduced in the presence of daidzein (0.02-0.1 mM) in a dose-dependent manner. High glucose levels induced the overexpression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB proteins in HUVECs, which was suppressed by treatment with 0.04 mM daidzein. These findings indicate the potential of daidzein to reduce high glucose-induced oxidative stress.