The aim of this study was to investigate the effect of 17β-estradiol (E2) on hepatocyte apoptosis after reduced-size hepatic ischemia/reperfusion (I/R) injury and its mechanism. A rat model of reduced-size hepatic I/R injury was established. Sprague-Dawley rats were randomly allocated into sham, I/R, and E2 + I/R group. 17β-Estradiol (4 mg/kg) or the vehicle was administered i.p. 1 h before ischemia and immediately after operation. For each group, 10 rats were used to investigate the survival during a week after reperfusion. Blood samples and liver tissues were obtained in the remaining animals after 3, 6, 12, and 24 h of reperfusion to assess serum aspartate aminotransferase and alanine aminotransferase levels, liver tissue malondialdehyde concentration, superoxide dismutase activity, and histopathologic changes. Apoptosis ratio; expression of cytochrome c, Bcl-2, and Bax proteins; and enzymatic activities of caspase 9 and caspase 3 were performed in the samples at 12 h after reperfusion. The serum aspartate aminotransferase and alanine aminotransferase levels and tissue malondialdehyde concentration were increased in the I/R group, whereas the increase was significantly reduced by E2. The superoxide dismutase activity, depressed by I/R injury, was elevated back to normal levels by treatment with E2. Severe hepatic damage was observed by light microscopy in the I/R group, whereas administration of E2 resulted in tissue and cellular preservation. Furthermore, E2 inhibited hepatocellular apoptosis by upregulating the ratio of Bcl-2 and Bax expression, reduced cytosolic cytochrome c level, and decreased caspase 9 and caspase 3 activities. The 7-day survival rate was significantly higher in the E2 + I/R group than in the I/R group. These results indicated that E2 protects liver tissues from reduced-size hepatic I/R injury by suppressing mitochondrial apoptotic pathways.