Aim To explore the role of nuclear factor-κB (NF-κB) in high doses of glucose (HG) induced impairment of ECV304 cells, a vascular endothelial cell line. Methods Recombinant adenovirus mediated nuclear factor-KB supper-repres sor IκBαM with mutant IκBα was constructed. Western blot, electrophoretic mobility shift assay (EMSA) and reverse tran scriptase-polymerase chain reaction(RT-PCR) assay were applied in this study. Results HG induced IκBα degradation and nuclear factor-KB activation were found in ECV304 cells but not in ECV304/IκBαM cells infected with IκBαM recombinant adenovirus . The expression of inflammatory factors such as monocyte chemotactic protein-1 (MCP-1), endothelin-1 (ET-1) and intercellular adhesion molecule-1 (ICAM-1) of ECV304 cells incubated with HG was increased obviously, but ECV304/IκBαM cells could resist HG induced increase of the expression of inflammatory factors. Conclusions HG increase the expression of inflammatory factors in vascular endothelial cells. Inhibition of nuclear factor-κB activation can protect vascular endothelial cells from the cytotoxicity of HG.