Induction of antigen-specific tolerance is critical for autoimmunity prevention and immune tolerance maintenance. In addition to their classical role as sentinels of the immune response, dendritic cells (DCs) play important roles in maintaining peripheral tolerance through the induction/activation of regulatory T (T(reg)) cells. The possibility of generating tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Characterizing endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. We here report that the immunosuppressive neuropeptide vasoactive intestinal peptide (VIP) induces the generation of human tolerogenic DCs with the capacity to generate CD4 and CD8 T(reg) cells from their respective naive subsets. The presence of VIP during the early stages of DC differentiation from blood monocytes generates a population of IL-10-producing DCs unable to fully mature after the effects of inflammatory stimuli. CD4 T(reg) cells generated with VIP-differentiated DCs resemble the previously described Tr1 cells in terms of phenotype and cytokine profile. CD8 T(reg) cells generated with tolerogenic VIP DCs have increased numbers of IL-10-producing CD8(+)CD28(-)-CTLA4(+) T cells. CD4 and CD8 T(reg) cells primarily suppress antigen-specific T(H)1-mediated responses. Therefore, the possibility of generating or expanding ex vivo tolerogenic DC(VIPs) opens new therapeutic perspectives for treating autoimmune diseases and graft-versus-host disease after allogeneic transplantation in humans.