Here, Negishi et al. investigate the role of interferon-regulatory factor 3 (IRF3) in intestinal homeostasis. Irf3−/− mice showed more severe symptoms of dextran sulphate sodium-induced colitis and impaired disease recovery compared with control mice. Thymic stromal lymphopoietin (TSLP) and interleukin-33 (IL-33) have homeostatic functions in the gut, and their expression levels were lower in Irf3−/− mice than in wild-type controls, both in the steady state and during colitis. IRF3-dependent expression of TSLP and IL-33 was induced in response to faecal (possibly microbiota-derived) nucleic acids and involved signalling through one of the adaptor proteins MAVS and STING. Finally, IRF3 cooperated with nuclear factor-κB to promote Tslp transcription. Further molecular links between the intestinal microbiota and IRF3-dependent homeostasis remain to be identified.