Cholera toxin (CT) is an ADP-ribosylating bacterial exotoxin that has been used as an adjuvant in animal studies of oral immunization. The mechanisms of mucosal immunogenicity and adjuvanticity of CT remain to be established. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), which participates in the induction of immune tolerance, in CT-mediated breakdown of oral tolerance. When IDO-deficient (IDO-/-) mice and their littermates were given oral ovalbumin, significant changes in antibody responses, footpad swelling and CD4+ T cell proliferation were not observed in IDO-/- mice. Feeding of CT decreased IDO expression in mesenteric lymph nodes (MLN) and Peyer''s patch (PP). CT-induced downregulation of IDO expression was reversed by inhibitors of nuclear factor-kappa B (NF-κB), pyrrolidine dithiocarbamate and p50 small interfering RNA. IDO expression was downregulated by the NF-κB inducers lipopolysaccharide and tumor necrosis factor-α. CT dampened IDO activity and mRNA expression in dendritic cells from MLN and PP. These data indicate that CT disrupts oral tolerance by activating NF-κB, which in turn downregulates IDO expression. This study betters the understanding of the molecular mechanism underlying CT-mediated abrogation of oral tolerance.