Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR) might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS) or inhibited iNOS activity in the APC(Min/+)Msh2(-/-) mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APC(Min/+)Msh2(-/-) mice.