Interleukin (IL)-10-producing type 1 regulatory T (Tr1) cells, which are Foxp3⁻memory T lymphocytes, play important roles in peripheral immune tolerance. We investigated whether Tr1 cells exert immunoregulatory effects in a mouse model of acute graft-versus-host disease (GVHD). Mouse CD4⁺ T cells were induced to differentiate in vitro into Tr1 cells using vitamin D3 and dexamethasone, and these donor-derived Tr1 cells were infused on the day of bone marrow transplantation. The Tr1 cell-transferred group showed less weight-loss and a twofold higher survival rate than the GVHD group, together with markedly decreased histopathologic grades. It was associated with the expansion of CD4⁺IL-4⁺ type 2 T-helper (Th2) cells and CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells. Furthermore, Tr1 cells decreased the numbers of CD4⁺interferon-γ⁺ Th1 and CD4⁺IL-17⁺ Th17 cells. Recipient mice harbored some Foxp3⁺ Tregs due to adoptive transfer of Tr1 cells, together with the upregulated expression of costimulatory molecules, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and inducible T-cell costimulator (ICOS); however, the Treg cells did not show the plasticity. Therefore, adoptive Tr1 cell therapy may be effective against manifestations of GVHD, exert immunomodulatory effects in a manner dependent on CTLA-4 and ICOS, and induce differentiation of the transferred Tr1 cells into Foxp3⁺ Treg cells.