Objective To observe the effect of evodiamine on the injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) and investigate its impossible mechanism. Methods HUVECs were used to make the inflammatory injured cell model. The cultured cells in vitro were divided into a normal control group, LPS model group, DMSO control group, low- and high-dose evodiamine group. The cell survival rate was detected by CCK-8 assay. The content of nitric oxide (NO) in the medium serum was detected by ELISA. Nuclear factor-kappa Bp65 (NF-κBp65) mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). The protein expression of Toll-like receptor 4 (TLR4) and NF-κBp65 was detected by Western blotting. Results Compared with the control group, the cell survival rate and NO content obviously decreased, and the expression levels of TLR4 and NF-κBp65 increased in the LPS treated group. Compared with LPS group, the pre-treatment of evodiamine obviously improved the cell survival rate, promoted the secretion of NO, inhibited the expression of TLR4 and NF-κBp65 and down-regulated the mRNA expression of NF-κBp65. Conclusion Evodiamine can relieve the inflammatory injury of HUVECs induced by LPS and promote the synthesis of NO, which may be related to the regulation of TLR4/NF-κB signaling pathway.