Aim To investigate the protective effect of acetaminophen on the injury of human umbilical vein endothelial cells(HUVEC) induced by hydrogen peroxide in vitro and to explore its underlying mechanism. Methods The HUVEC line was subcultured in vitro and used for experiment.This study was conducted as follows: normal control group,hydrogen peroxide(H2O2)-injury group(0.1 mmol/L),acetaminophen protective group(25 μmol/L,50 μmol/L and 100 μmol/L) was pretreatmented for 1 hour.The cell viability was measured by MTT assay.The content of malondialdehyde(MDA) and super oxide dismutase(SOD) was determined by kit.The expression of apoptosis-related protein caspase-3 was determined by Western blotting.Apoptosis of the cells was analyzed by flow cytometry. Results Comparied with normal control group,H2O2 can obviously damage vascular endothelial cells(P<0.05).It shows as the decrease of viability of HUVEC,the increase of the content of MDA,the decrease of SOD activity,the increase of caspase-3 expression and apoptosis rate.Acetaminophen can disturb the damage of H2O2-induced endothlial celles.It can increase viability of HUVEC,decrease the content of MDA,increase SOD activity,decrease caspase-3 expression and apoptosis rate.The effect of acetaminophen showed a dose-dependent manner(P<0.05). Conclusions The results show that acetaminophen can decrease the damage of cultured vascular endothelial celles injured by H2O2.It also can inhibit the apoptosis of vascular endothelial cells induced by H2O2.The protective effect of acetaminophen may relate with antioxidation and inhibiting pro-apoptotic protein caspase-3.