BACKGROUND: Many scholars attempt to xenotransplantation because of shortage of human donor hepatocytes. In the field of hepatocyte xenotransplantation, two different mammalian species, i.e., pig-to-rat, and pig-to-rodent, are much reported. Hepatocyte transplantation between two different classes has been rarely reported. OBJECTIVE: To investigate the mechanisms of cell-mediated immune response in hepatocytes xenotransplantation between tilapia and rat. METHODS: The tilapia (donor) hepatocytes were isolated by collagenase cold digestion and adjusted to 2×107 /mL using physiological saline. Thirty-six SD (recipient) rats were randomly divided into transplantation and control groups (n = 18). Rats from the control group received injection of physiological saline into the spleen, and those from the transplantation group received injection of tilapia hepatocytes. At each time point (4 hours, 8 hours, 24 hours, 3 days, and 5 days) after transplantation, two rats were respectively sacrificed for histological changes examination using hematoxylin-eosin staining and detection of CD4+/CD8+ lymphocytes surrounding the grafts using immunohistochemical SABC method. RESULTS AND CONCLUSION: The transplanted hepatocytes were rejected a few hours after transplantation. Surviving hepatocytes in spleens were hardly seen 8 hours after transplantation. The aggregation of CD4+CD8+ lymphocytes was detected surrounding the grafts 4 hours after transplantation. These findings suggest that tilapia hepatocytes transplantation into rat spleen induces rejection within a few hours, cell-mediated immune response is involved in the rejection of hepatocytes xenotransplantation, and natural killer cells may play an important role in this kind of rejection.