Notch signaling pathway has been demonstrated to mediate radioresistance of several tumors. Our study aims to explore the function of Notch1/HES1 pathway in the radioresistance of colorectal cancer (CRC). The results demonstrated that expressions of Notch1 and Hes1 were up-regulated with the increasing irradiation dose. DAPT (N-[(3,5-difluorophenacetyl)acety1]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester) or si-Notch1 reduced expressions of Notch1 and Hes1, exacerbated irradiation-induced cell proliferation inhibition, and improved radiosensitivity of CRC cells. Moreover, DAPT or si-Notch1 increased radiation-induced DNA damage and attenuated radiation-triggered DNA-PK activity. Furthermore, xenograft in nude mice demonstrated that co-treated with DAPT and irradiation could inhibited tumor growth additively in vivo. Taken together, inhibition of Notch1/Hes1 signaling pathway enhances radiosensitivity of CRC cells, providing a potential therapeutic target to improve the therapeutic effect of radiotherapy for CRC patients.