CD4CD25 regulatory T cells (Treg) are potent immunosuppressive cells active in controlling normal pathological immune responses. The mechanisms of this suppression have been investigated under various conditions. In this report, tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/death receptor 5 (DR5) was explored as one of the pivotal factors for the suppression and cytotoxicity induced by CD4CD25 Treg. Cell death was involved in the suppression induced by activated CD4CD25 Treg in vitro. The induction of CD4 T cell death was not mediated by the CD95/CD95L pathway, but rather depended upon the upregulation of TRAIL in the Treg. Blocking the TRAIL/DR5 pathway resulted in a significant reduction of the suppressive activity as well as the cytotoxic effects of Treg in vitro. Activated Treg displayed TRAIL-dependent cytotoxicity against CD4 T cells in vivo. The prolonged survival of allogeneic skin grafts induced by Treg was inhibited by DR5-blocking antibodies. Our findings suggest that the TRAIL/DR5 pathway is one of the mechanisms used by Treg to regulate immune responses both in vitro and in vivo.