Xeno-hepatocyte transplantation has the possibility to substitute for clinical liver transplantation in certain restricted hepatic diseases such as inherited metabolic disorders. To overcome human complement-dependent cytotoxicity on xeno-hepatocytes, the effectiveness of ex vivo transfer with the homologous restriction factor 20 (HRF20, CD59) gene was examined on primary-cultured xeno-hepatocytes using a retroviral vector. Primary-cultured rat hepatocytes transduced with HRF20 cDNA expressed HRF20 antigen by flowcytometric analysis and showed the integration of HRF20 cDNA to the genomic DNA by the polymerase chain reaction. The viability of rat hepatocytes incubated with 50% human serum was decreased due to complement-dependent cytotoxicity, whereas that of the transfectant was significantly protected (77.2 ± 9.4 vs. 97.8 ± 5.2%, p < 0.01). It was concluded that primary-cultured xeno-hepatocytes transduced with HRF20 cDNA using a retroviral vector could escape complement-dependent cytolysis by human serum.