The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-β (IFN-β) production, which protected mice from experimental colitis. The LAB-induced IFN-β response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-β than LAB, and dsRNA was not involved in pathogen-induced IFN-β induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatory and protective immune responses.