Fed protein undergoes processing and coupling to major histocompatibility complex (MHC) II molecules during passage through the intestinal epithelium, generating a tolerogenic form of the antigen in serum. Transfer of this factor to naïve animals induces tolerance in the recipient. In this study, we investigate what impact colonization with Gram-positive (Lactobacillus plantarum) or Gram-negative (Escherichia coli) bacteria has on tolerogenic processing in the gut. Germ-free (GF), monocolonized or conventional mice were fed ovalbumin (OVA), and their serum was collected and transferred to naïve conventional recipients that were tested for delayed-type hypersensitivity against OVA after parenteral immunization. A transferable tolerogenic factor was produced by conventional mice, but not by mice that were germ free or monocolonized with either E. coli or L. plantarum. Conventional, but neither GF nor monocolonized mice showed upregulation of MHCII expression in the epithelium of small intestine. The results suggest that a complex intestinal microflora is needed to support oral tolerance development.