Colorectal cancer is a leading cause of cancer-related deaths. Mutations in the innate immune sensor AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. Here, we found that Aim2-deficient mice were hypersusceptible to colonic tumor development. Production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in Aim2-deficient mice; however, intestinal stem cells were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2. Susceptibility of Aim2-deficient mice to colorectal tumorigenesis was enhanced by a dysbiotic gut microbiota, which was reduced by reciprocal exchange of gut microbiota with healthy wild-type mice. These findings uncover a synergy between a specific host genetic factor and gut microbiota in determining the susceptibility to colorectal cancer. Therapeutic modulation of AIM2 expression and microbiota has the potential to prevent colorectal cancer. Display omitted •AIM2 is required to mediate protection against colorectal cancer•AIM2 suppresses overt proliferation in enterocytes•AIM2 inhibits expansion of the intestinal stem cell population•Transfer of healthy microbiota dampens tumor development in Aim2-deficient mice The cytosolic DNA sensor AIM2 regulates stem cell proliferation in the intestinal mucosa in an inflammasome-independent fashion, contributing to a decrease in the likelihood of colorectal cancer development.