Xenotransplantation of hepatocytes appears to be a novel promising therapy for some forms of liver disease, and may well overcome the problem of donor shortage. We have previously reported that hepatocytes with a spheroidal shape (spheroids) are ideal for cell transplantation. The application of gene transfer techniques to this hepatocyte transplantation could possibly regulate the xenogeneic rejection reaction and, therefore, result in prolongation of the survival of the transplanted hepatocytes. In this study, we chose the adenovirus as a vector and an immunosuppressive cytokine named viral IL-10 (vIL-10) for transfection. A series of experiments was performed to elucidate the efficacy of transfection to the spheroids with adenovirus vectors and the effect of transfected vIL-10 on the survival of xenogeneic hepatocytes. We examined the cell survival quantitatively by evaluating beta-galactosidase (beta-gal) activity, which was transfected into the hepatocytes in the xenogeneic spleen, and semiquantitatively by the histological findings. The results of in-vitro studies identified an efficient expression of the beta-gal gene within the spheroids infected with Ad-CMVLacZ (LacZ-encoding adenovirus vector with CMV promotor) and the presence of BCRF1 mRNA within the spheroids transfected with AdCMVvIL-10 (vIL-10-expressing adenovirus vector with CMV promotor) under the condition of 1 MOI, for 1 h. Xenogeneic hepatocytes with a spheroidal shape showed comparable survival to syngeneic hepatocytes for up to 4 days after transplantation with co-transplantation of the vIL-10-transfected hepatocytes. From this study, we concluded that adenovirus-mediated vIL-10 gene transfer prolongs the survival of xenogeneic hepatocyte spheroids. Furthermore, spheroids possess ideal properties for gene transfection, as well as cell transplantation.