Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9-sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3- proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3- Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-β but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.