© 2018 American Academy of Allergy, Asthma & Immunology Background: Exposure to dietary constituents through the mucosal surface of the gastrointestinal tract generates oral tolerance that prevents deleterious T cell–mediated immunity. Although oral tolerance is an active process that involves emergence of CD4+ forkhead box p3 (Foxp3)+ regulatory T (Treg) cells in gut-associated lymphoid tissues (GALTs) for suppression of effector T (Teff) cells, how antigen-presenting cells initiate this process remains unclear. Objective: We sought to determine the role of plasmacytoid dendritic cells (pDCs), which are known as unconventional antigen-presenting cells, in establishment of oral tolerance. Methods: GALT-associated pDCs in wild-type mice were examined for their ability to induce differentiation of CD4+ Teff cells and CD4+Foxp3+ Treg cells in vitro. Wild-type and pDC-ablated mice were fed oral antigen to compare their intestinal generation of CD4+Foxp3+ Treg cells and induction of oral tolerance to protect against Teff cell–mediated allergic inflammation. Results: GALT-associated pDCs preferentially generate CD4+Foxp3+ Treg cells rather than CD4+ Teff cells, and such generation requires an autocrine loop of TGF-β for its robust production. A deficiency of pDCs abrogates antigen-specific de novo generation of CD4+Foxp3+ Treg cells occurring in GALT after antigenic feeding. Furthermore, the absence of pDCs impairs development of oral tolerance, which ameliorates the progression of delayed-type hypersensitivity and systemic anaphylaxis, as well as allergic asthma, accompanied by an enhanced antigen-specific CD4+ Teff cell response and antibody production. Conclusion: pDCs are required for establishing oral tolerance to prevent undesirable allergic responses, and they might serve a key role in maintaining gastrointestinal immune homeostasis.