Tolerogenic dendritic cells (tDCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tDCs. In this article, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tDCs. The VIP/PACAP-generated DCs are CD11clowCD45RBhigh, do not upregulate CD80, CD86, and CD40 following lipopolysaccharide (LPS) stimulation, and secrete high amounts of IL-10. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. VIP/DCs induce antigen-specific tolerance in vivo, suppress delayed-type hypersensitivity (DTH), and T cells from VIP/DC-inoculated mice transfer the suppression to naïve hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites that exhibit immune deviation or privilege.