Aim To study the effects of 7-difluoromethyl-5,4′-dimethoxygenistein (dFMGEN) on the apoptosis to H₂O₂-induced vascular endothelial cells and its molecular mechanism. Methods The oxidative stress injury model was established by human umbilical vein endothelial cells induced by 1 mmol/L H₂O₂. The experiment was divided into blank control group, H₂O₂ group, vehicle group, 100 μmol/L genistein group and different concentrations (0.1,0.3,1, 3,0 μmol/L) dFMGEN groups. The reactive oxygen species (ROS) generation was examined by DCFH-DA activated fluorescence flow cytometry (FCM). Apoptotic morphology was observed by acridine orange staining under fluorescence microscopy. The apoptotic rate was detected by PI staining FCM. The expression of Caspase-3 was determined by Western blot. Results When vascular endothelial cells were treated with 1 mmol/L H₂O₂ for 24 h, the generation of ROS was highly added, obvious morphological changes of apoptosis was found by acridine orange staining under fluorescence microscopy, the apoptotic rate was increased and the expression of Caspase-3 was up-regulated. After pretreating with dFMGEN, results showed that dFMGEN could reduce the release of ROS, decrease the apoptotic rate and down-regulate the expression of Caspase-3. Conclusion dFMGEN can effectually inhibit the apoptosis of vascular endothelial cells induced by H₂O₂, which is probably due to the decrease of ROS generation and downregulation of Caspase-3, thereby inhibiting apoptosis of vascular endothelial cells induced by H₂O₂.