Abstract: Objective:To determine the optimal enzymatic hydrolysis conditions of polypeptides from Apostichopus japonicas and explore the protective effect of enzymatic hydrolysate against hydrogen peroxide(H₂O₂)induced oxidative stress in human vascular endothelial cell line(EA.hy926). Methods:The single factor methodology was carried out to determine the optimal temperature,enzyme volume and pH. The response surface methodology was used to optimize the enzymatic hydrolysis conditions. The in vitro antioxidant activities of enzymolysis polypeptide were investigated. The MTS method was performed to evaluated the protective effects of enzymatic hydrolysate against H₂O₂ induced oxidative stress in EA.hy926. The MDA and SOD values were monitored. Results:Animal protease was the most suitable protease.The optimum conditions of enzymatic hydrolysis were as follows:Solid-liquid ratio was 1:20 g/mL,the enzymatic hydrolysis time was 2 h,the enzymatic hydrolysis temperature was 50℃,the enzyme concentration was 7000 U/g,and the pH was 7.5.Under these conditions,the DPPH free radical scavenging rate of the peptides was 68.81%,and the relative error was 1% with the predicted value of the model(68.35%),which indicated the regression model was reliable. The IC₅₀ inhibitory concentrations of DPPH radical,hydroxyl radical(·OH),superoxide anion radical(O₂^-·)and ABTS⁺·were 9.01,0.63,10.89 and 20.53 mg/mL respectively,which showed that the enzymolysis polypeptide had good antioxidant activity in vitro. A cell injury model was established with a concentration of 200 μmol/L H₂O₂.Compared with H₂O₂ group,the antioxidant peptides of middle and high dose groups could significantly inhibit oxidative damage of vascular endothelial cells induced by H₂O₂,reduce MDA content and increase SOD activity. Conclusion:The antioxidant peptides prepared by enzymatic hydrolysis of animal proteolytic enzymes had significant protective effect on human umbilical vein endothelial cells EA. hy926 and had a significant dose-effect relationship.