Mesenchymal stem cells (MSCs) have been considered to be an ideal cellular source for graft-versus-host disease (GVHD) treatment due to their unique properties, including tissue repair and major histocompatibility complex (MHC)-unmatched immunosuppression. However, preclinical and clinical data have suggested that the immunomodulatory activity of MSCs is not as effective as previously expected. This study was performed to investigate whether the immunomodulatory capacity of MSCs could be enhanced by combination infusion of regulatory T (Treg) cells to prevent acute GVHD (aGVHD) following MHC-mismatched bone marrow transplantation (BMT). For GVHD induction, lethally irradiated BALB/c (H-2(d)) mice were transplanted with bone marrow cells (BMCs) and spleen cells of C57BL/6 (H-2(b)) mice. Recipients were injected with cultured recipient-derived MSCs, Treg cells, or MSCs plus Treg cells (BMT + day 0, 4). Systemic infusion of MSCs plus Treg cells improved clinicopathological manifestations and survival in the aGVHD model. Culture of MSCs plus Treg cells increased the population of Foxp3(+) Treg cells and suppressed alloreactive T-cell proliferation in vitro. These therapeutic effects were associated with more rapid expansion of donor-type CD4(+)CD25(+)Foxp3(+) Treg cells and CD4(+)IL-4(+) type 2 T-helper (Th2) cells in the early posttransplant period. Furthermore, MSCs plus Treg cells regulated CD4(+)IL-17(+) Th17 cells, as well as CD4(+)IFN-γ(+) Th1 cells. These data suggest that the combination therapy with MSCs plus Treg cells may have cooperative effects in enhancing the immunomodulatory activity of MSCs and Treg cells in aGVHD. This may lead to development of new therapeutic approaches to clinical allogeneic hematopoietic cell transplantation.