The aim of this study was to determine if estrogen plays any role in the process of hepatic regeneration of nonligated lobe after portal vein branch ligation (PBL). We also investigated whether estrogen has any association with serotonin action during liver regeneration. Ovariectomized female rats with (E group) or without (non-E group) estrogen pellet were subjected to PBL on the left and middle lobes. Thereafter, the rats were killed, and blood, liver, and small intestine were sampled and analyzed. Sham animals underwent only ovariectomy and laparotomy. The E group showed a significantly greater regeneration rate than the non-E group at days 1, 2, and 7 after PBL. The activation of hepatic regeneration-related genes (such as IL-6, TNF-α, hepatic growth factor, c-fos, and c-myc) was also significantly higher in the E group as compared with the non-E group. Gene expression of serotonin receptor (5-HT2A) in the liver and tryptophan hydroxylase 1 in the small intestine were also up-regulated in the E group, indicating an activation of serotonin system in the E group. Additionally, total intestinal flow, portal venous flow, and hepatic arterial flow determined by fluorescent microsphere were significantly higher in the E group compared with the non-E group. Moreover, serotonin receptor antagonist (ketanserin) significantly attenuated liver regeneration rate in the E group. These results indicated that estrogen plays an important role in the process of liver regeneration after PBL. Our results also indicated that estrogen is at least partly related to the activation of serotonin system, which is also important in the process of liver regeneration.