Significance Intestinal barrier plays a key role in maintaining organismal health. Here we find cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) deficiency compromises intestinal epithelial barrier and exacerbates inflammation. cGAS-deficient mice were highly susceptible to colitis-associated colon cancer (CAC) but not sporadic colon cancer. Surprisingly, the role of cGAS in this process appears to be independent of stimulator of interferon genes (STING)-induced type I interferon signaling, because mice lacking STING or type I interferon receptor were less susceptible to CAC than those lacking cGAS. cGAS but not STING is highly expressed in intestinal stem cells. These results suggest that cGAS has a unique role in protecting the intestinal epithelial barrier and preventing colon cancer development.