Although cell sheet technology has recently been developed for use in both animal experiments and in the clinical setting, it remains unclear whether transplanted hepatocyte sheets improve the liver function in vivo. Radiation-induced liver damage (RILD) combined with partial hepatectomy (PH) has been reported to suppress the proliferation of host hepatocytes and induce critical liver failure. The aim of this study was to improve the liver function in the above-mentioned diseased rat model (RILD+PH) using multilayered hepatocyte sheet transplantation. In this study, we used Fischer rats as a donor for primary hepatocytes and dermal fibroblast isolation. Cocultured multilayered hepatocyte sheets were generated by disseminating hepatocytes onto fibroblasts cultured beforehand on temperature-responsive culture dishes. Four cell sheets were transplanted into the recipient rats subcutaneously. Prior to transplantation, RILD (50 Gy) with 2/3PH was induced in the recipients. The same model was applied in the control group without transplantation. The serum was collected each week. The rats in both groups were sacrificed at 2 months after transplantation for the histological analysis. Consequently, the serum albumin concentrations were significantly higher in the transplant group than in the control group (54.3 +/- 9.6 vs. 32.7 +/- 5.7 mg/ml; p < 0.01) after 2 months and comparable to the serum albumin levels in the normal rats (58.1 +/- 6.4 mg/ml). In addition, treatment with the transplanted sheets significantly improved the survival rate (57% vs. 22%, p<0.05), and the hepatocyte sheets showed the storage of albumin, glycogen, and bile canaliculus structures. Some hepatocytes and fibroblasts were positive for Ki-67, and vascularization was observed around the cell sheets. Transplanted multilayered hepatocyte sheets can survive with additional proliferative activity, thereby maintaining the liver function in vivo for at least 2 months, providing metabolic support for rats with RILD.