OBJECTIVE Establishing an effective humoral immunity is an important host defense mechanism in intestinal mucosa. T follicular helper (Tfh) cells are a spectrum of CXCR5 expressing T helper cells that induce antigen-specific B cell differentiation. Because the differentiation of T helper cells is largely regulated by innate immunity, we addressed whether TRIF signaling regulates Tfh cell differentiation and its ability to trigger humoral immune responses in the intestine. METHOD CD4+CXCR5+ T cells, B cells, and plasma cells in the Peyer's patches (PPs) of WT and TRIF-deficient (TrifLPS2) mice were analyzed by flow cytometry at the baseline, 9 days post primary infection, and 7 days post-secondary infection with Y. enterocolitica. Y. enterocolitica-specific CD4+CXCR5+ T cells were generated in vitro by co-culturing peritoneal macrophages with splenic naïve T cells in the presence of Y. enterocolitica lysate. WT and TrifLPS2 mice received CD4+CXCR5+ T cells isolated either from Y. enterocolitica-primed WT mice or generated in vitro. These mice were infected with Y. enterocolitica and followed up to 4 weeks. Y. enterocolitica-specific IgA and IgG were measured in stool and serum samples, respectively. RESULTS At baseline, CD4+CXCR5+ T cell proportion was higher but the proportion of B cells and plasma cells was lower in the PPs of TrifLPS2 mice compared to WT mice. After infection, the proportion of plasma cells also became higher in the PPs of TrifLPS2 mice compared to WT mice. Corresponding increase of Y. enterocolitica-specific stool IgA but not serum IgG was found in TrifLPS2 mice compared to WT mice. Both in vivo isolated and in vitro generated CD4+CXCR5+ T cells induced protective immunity against Y. enterocolitica infection. CONCLUSION Our results reveal a novel role of TRIF in the regulation of humoral immunity in the intestine that can be utilized as a basis for a unique vaccine strategy.