This study investigates the influence of 17beta-estradiol (E2) on hydrogen peroxide (H2O2)-induced human vascular endothelial cell (HUVEC) senescence. HUVECs were divided into four groups, namely control group, H2O2 stimulation group, E2 intervention group and ICI182780 (ICI) intervention group. The aging-related β-galactosidase activities, cytochrome C oxidase activities, intracellular ATP levels, intracellular reactive oxygen species (ROS) levels and phosphorylated Rb protein expressions were mainly observed. Of which, senescence-associated β-galactosidase activities were detected using immunohistochemical staining, cytochrome C oxidase activities and intracellular ATP levels were detected using commercial kits, ROS levels were detected by fluorescence microscopy and fluorescence microplate reader, immunoblotting was used to quantitatively detect the expressions of phosphorylated Rb proteins. After continuous treatment of H2O2, the senescent phenotypes appeared in the HUVECs. The percentage of positive SA-βgal staining cells and the phosphorylated Rb expressions were significantly increased; intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels were elevated. Compared with the H2O2 stimulation group, E2 intervention significantly decreased the positive rate of SA-β-gal staining, the phosphorylated Rb protein levels, the intracellular ROS levels, cytochrome C oxidase activities and intracellular ATP levels. Pretreatment of estrogen receptor blocker ICI182780 weakened the role of E2. These results indicated that H2O2 could induce HUVEC senescence; 17beta-E2 might relieve H2O2-induced mitochondrial damage through estrogen receptor and delay the vascular endothelial cell senescence.