Acute graft-versus-host disease (aGvHD) is a frequent complication after allogeneic bone marrow/stem cell transplantation (BMT/SCT) induced by co-transplanted alloreactive conventional donor T cells. We previously demonstrated that the adoptive transfer of donor CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) at the time of BMT prevents aGvHD in murine models. Yet, the therapeutic potential of donor Treg for the treatment of established aGvHD has not yet been studied in detail. We now used in vitro expanded phenotypically and functionally stable murine Treg to explore their therapeutic efficacy in haploidentical aGvHD models. Upon transfer donor Treg ameliorate clinical and histologic signs of aGvHD and significantly improve survival. They migrate to lymphoid as well as aGvHD target organs, predominantly the gastrointestinal tract, where they inhibit the proliferation of conventional T cells, reduce the influx of myeloid cells, and the accumulation of inflammatory cytokines. Successfully treated animals restore aGvHD-induced tissue damage in target organs and lymphoid tissues, thereby supporting lymphocyte reconstitution. The therapeutically applied Treg population survives long term without conversion into pathogenic effector T cells. These results demonstrate that donor Treg not only prevent aGvHD, but are also efficacious for the treatment of this life-threatening BMT complication.