Acute graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT) for the treatment of leukemia and other immunogenetic disorders. The use of tolerogenic dendritic cells (DCs) with potent immunoregulatory properties by inducing the generation/activation of regulatory T cells (Tr) for the treatment of acute GVHD following allogeneic BMT has been recently established. Here we report the use of the known immunosuppressive neuropeptide, vasoactive intestinal polypeptide (VIP), as a new approach to inducing tolerogenic DCs with the capacity to prevent acute GVHD. DCs differentiated with VIP impair allogeneic haplotype-specific responses of donor CD4+ T cells in transplanted mice by inducing the generation of Tr in the graft. Importantly, VIP-induced tolerogenic DCs did not abrogate the graft versus leukemia response, probably because they do not abrogate cytotoxicity of transplanted T cells against the leukemic cells. Therefore, the inclusion of VIP-induced tolerogenic DC in future therapeutic regimens may facilitate the successful transplantation from mismatched donors, reducing the deleterious consequences of acute GVHD, extending the applicability of BMT.