Intestinal microbes promote the injurious effects of radiation on those tissues. However, the molecular factors mediating this effect are largely unknown. In this work, we explored the effects of orally administered antibiotics and MyD88, a key adapter molecule in toll-like receptor signaling, on molecular and cellular responses of mouse colon to radiation. Results show that oral antibiotics lowered radiation-induced colonic damage by protecting epithelial cells against radiation-induced apoptosis, leading to increased survival of crypts. MyD88 deficiency partially phenocopied the effects of oral antibiotics on apoptosis and crypt survival, suggesting that colonic microbes exert their injurious effects in part via that molecule. Analysis of DNA double-strand breaks, the primary genotoxic lesions induced by radiation, showed that their early induction in mouse colon was unaffected by MyD88. However, MyD88 deficiency resulted in the later disappearance of DNA double-strand breaks. Loss of DNA double-strand breaks was accompanied by the evidence of increased activation of both the non-homologous end-joining and homologous recombination pathways of DNA repair in MyD88-deficient mice. These results show that colonic microbes and MyD88 regulate DNA double-strand break repair in irradiated mouse colon, effects which exert significant control over radiation-induced apoptosis and crypt survival.