Microbial pattern molecules in the intestine play immunoregulatory roles via diverse pattern recognition receptors. However, the role of the cytosolic DNA sensor AIM2 in the maintenance of intestinal homeostasis is unknown. Here, we show that Aim2−/− mice are highly susceptible to dextran sodium sulfate-induced colitis that is associated with microbial dysbiosis as represented by higher colonic burden of commensal Escherichia coli. Colonization of germ-free mice with Aim2−/− mouse microbiota leads to higher colitis susceptibility. In-depth investigation of AIM2-mediated host defense responses reveals that caspase-1 activation and IL-1β and IL-18 production are compromised in Aim2−/− mouse colons, consistent with defective inflammasome function. Moreover, IL-18 infusion reduces E. coli burden as well as colitis susceptibility in Aim2−/− mice. Altered microbiota in inflammasome-defective mice correlate with reduced expression of several antimicrobial peptides in intestinal epithelial cells. Together, these findings implicate DNA sensing by AIM2 as a regulatory mechanism for maintaining intestinal homeostasis. Display omitted •AIM2 senses intestinal microbial DNA and activates the inflammasome•The AIM2 inflammasome protects mice from experimental colitis•The AIM2 inflammasome controls the growth of commensal Escherichia coli•The inflammasome triggers antimicrobial peptide production by epithelial cells AIM2 is a cytosolic DNA sensor. Hu et al. demonstrate that intestinal microbial DNA activates the AIM2 inflammasome. AIM2 activation leads to the production of IL-1β and IL-18, which participate in the regulation of intestinal microbiota such as Escherichia coli via induction of the antimicrobial peptides in intestinal epithelial cells.