The role of symbiotic bacteria in the development of antigen-specific immunity remains poorly understood. Previous studies showed that sensing of symbiotic bacteria by nucleotide-binding oligomerization domain-containing protein 2 (Nod2) regulates antibody responses in response to nasal immunization with antigen and cholera toxin (CT). In this study, we examined the role of the microbiota in the adjuvant activity of CT induced after oral immunization with antigen. Germ-free (GF) mice showed impaired production of antibody responses and T-cell-specific cytokines after oral immunization when compared with that observed in conventionally raised mice. Similar to GF mice, Nod2-deficient mice showed reduced humoral responses upon oral immunization with antigen and CT. Treatment with CT enhanced the production of interleukin-1 (IL-1), but not tumor necrosis factor- or IL-12p40, induced by stimulation of dendritic cells with muramyl dipeptide, the Nod2 ligand. Mechanistically, the enhanced production of IL-1 induced by muramyl dipeptide and CT stimulation required Nod2 and was mediated by both increased synthesis of pro-IL-1 and caspase-1 activation. Furthermore, antigen-specific antibody and cytokine responses induced by CT were impaired in orally immunized IL-1-deficient mice. Collectively, our results indicate that Nod2 stimulation by symbiotic bacteria contributes to optimal CT-mediated antigen-specific oral vaccination through the induction of IL-1 production. 2019 John Wiley & Sons Ltd.