Tolerogenic dendritic cells (tDCs) potently induce and maintain tolerance based on their distinct characteristics compared with conventional DCs. Recent reports show that donor or host tDCs promote allograft survival in mice. In this study, the efficacy of third-party tDCs in the prevention of acute graft-versus-host disease (aGVHD) was evaluated. In vitro, tDCs derived from the bone marrow (BM) of D1 mice were induced by GM-CSF, IL-10 and TGF-β1. The phenotypes, expression of cytokines and suppression of tDCs were analysed. In vivo, the effects of adoptive transfer of third-party-tDCs were evaluated in an MHC-mismatched aGVHD mouse model. Survival, body weight, GVHD scoring, histopathological specimens and serum cytokines were analysed in tDC-treated mice and untreated controls. Tolerogenic DCs had low expression of MHC and co-stimulatory molecules, expressed high levels of ‘immunosuppressive’ cytokines and suppressed allo-CD4+T cell proliferation. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Fortunately, third-party tDCs transferred at low doses (104) effectively prolonged survival after allo-BMT. Furthermore, in the mice treated with 104 tDCs, serum levels of IL-10/TGF-β were significantly higher and the percentage of Foxp3+ cells continually increased compared with the mice treated with other doses of tDCs. Third-party tDCs play a crucial role in reducing the severity of aGVHD by modulating the secretion of various cytokines and expanding Foxp3+ regulatory T cells, which suggests the possibility of using third-party tDCs for therapeutic applications. Furthermore, special attention should be paid to the optimal range of tDCs for preventing allograft rejection.