Experimental colitis is mediated by inflammatory or dysregulated immune responses to microbial factors of the gastrointestinal tract. In this study we observed that administration of Toll-like receptor 9 (TLR9) agonists suppressed the severity of experimental colitis in RAG1–/–but not in SCID mice. This differential responsiveness between phenotypically similar but genetically distinct animals was related to a partial blockade in TLR9 signaling and defective production of type I IFN (ie, IFN-α/β) in SCID mice upon TLR9 stimulation. The addition of neutralization antibodies against type I IFN abolished the antiinflammatory effects induced by TLR9 agonists, whereas the administration of recombinant IFN-β mimicked the antiinflammatory effects induced by TLR9 agonists in this model. Furthermore, mice deficient in the IFN-α/β receptor exhibited more severe colitis than wild-type mice did upon induction of experimental colitis. These results indicate that TLR9-triggered type I IFN has antiinflammatory functions in colitis. They also underscore the important protective role of type I IFN in intestinal homeostasis and suggest that strategies to modulate innate immunity may be of therapeutic value for the treatment of intestinal inflammatory conditions.