Epstein-Barr virus-induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4+ T cells had reduced IFN-gamma production and failed to induce T cell-dependent colitis in mice. Similarly reduced IFN-gamma production was observed in vitro in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23R alpha, but not another IL-23R subunit, IL-12R beta 1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23R alpha expression via binding to the chaperone molecule calnexin and to IL-23R alpha in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23R alpha expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23R alpha variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23R alpha protein expression via calnexin under inflammatory conditions.