In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction. Liver Transpl 12:1112–1118, 2006. © 2006 AASLD.