Hepatocyte transplantation offers an alternative therapeutic approach in the treatment of liver-related diseases. Hemophilia B is a bleeding disorder lacking factor IX (FIX) production in the liver, and achieving more than 1% coagulation activity results in significant improvement in the quality of life of the patients. The aim of this study was to investigate the efficacy of hepatocyte transplantation in the mouse model of hemophilia B. We transplanted isolated normal mouse hepatocytes into the liver of FIX knock-out mice. In some recipient mice, additional hepatocyte transplantations were performed 15 days after the first transplant. The recipient plasma FIX activities increased at 1% to 2% and persisted throughout the experimental period. An additional increase was achieved by the repeated transplantation. Close correlation between FIX messenger RNA levels of the liver and plasma FIX activity levels was observed. These results demonstrate that hepatocyte transplantation can provide therapeutic benefits in the treatment of hemophilia B.