Abstract: The commensal microflora of the intestinal tract confer multiple health benefits to the host, including amelioration of inflammatory bowel disease (IBD). Yet, the exact mechanisms by which it ameliorates experimental colitis in animals and human IBD are largely unknown. We tested whether the attenuation of experimental colitis by probiotic bacteria is mediated by toll-like receptor (TLR) signaling. The severity of colitis was attenuated by delivery of nonviable, γ-irradiated, or by viable probiotics, but not by heat-killed probiotics, in wild-type mice in mice deficient in TLR2 or TLR4. In contrast we did not observe any inhibition of experimental colitis by probiotics, in mice deficient in MyD88 or TLR9. Furthermore, administration of probiotic DNA ameliorated the severity of experimental colitis, whereas methylated probiotic DNA, calf thymus DNA, and Dnase-treated probiotics had no effect. In subsequent studies, we identified that TLR9-induced type 1 IFN mediates the anti-inflammatory effects in experimental colitis. The addition of neutralization antibodies to type 1 IFN abolished the anti-inflammatory effects, whereas the administration of recombinant IFN-β mimicked the anti-inflammatory effects induced by TLR9 agonists.Taken together, these results indicate that the protective effects of probiotics are mainly mediated by their own DNA rather than by their metabolites or their ability to colonize the colon. These findings underscore the diverse effects of indigenous microbial TLR ligands in intestinal homeostasis and intestinal inflammation and suggest that strategies, that modulate type 1 IFN may be of therapeutic value for intestinal inflammatory conditions.