Background & Aims: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. Methods: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-pσ1), pσ1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4+) T-cell responses were determined. Results: A low dose of OVA-pσ1 reduced anti-OVA Ab and CD4+ T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-Ad tetramer staining showed that the numbers of OVA-specific CD4+ T cells were significantly reduced in lamina propria of mice fed OVA-pσ1 than those fed pσ1 only or PBS only. In fact, Foxp3 expressing CD25+ CD4+ T cells were markedly increased in this tissue. Nonetheless, CD25+ CD4+ T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) production compared with nontolerized mice. Conclusions: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4+ T cells and increased levels of TGF-β1 and IL-10 producing, CD25+ CD4+ regulatory T cells in both systemic and mucosal lymphoid tissues. © 2008 AGA Institute.