In order to investigate the role of Toll-like recepter 4(TLR4) in lipopolysaccharide(LPS)-induced NF-κB activation in human endothelial cells,LPS-stimulated ECV-304 cells were used as experimental model and the expression of TLR4 and effect of LPS on the expression were analysed with RT-PCR and Western blot assay. Moreover, the role of TLR4 in LPS-induced NF-κB activation in endothelial cells was explored with gene transfection of non-signaling mutant forms of TLR4 and anti-TLR4 monoclonal antibody. The results showed that LPS could upregulat the expression of TLR4 in time and dose-depentent manner and that transfection of non-signaling mutant forms of TLR4 and anti-TLR4 monoclonal antibody inhibited LPS-induced NF-κB activation in human endothelial cells obviously.These data indicated that TLR4 mediates LPS-induced NF-κB activation in human endothelial cells and it may play important role in endothelial cell activation and injury induced by LPS.