Abstract <p>Radiation therapy (RT) is one of the most important strategies for killing cancer cells and shrinking tumors. However, in colorectal cancer, the application of radiotherapy is restricted due to radioresistance. Therefore, exploring the detailed mechanisms of radioresistance may improve patient responses to irradiation and enhance survival rates. Here, we selected SW480 and SW620 cells for X-ray radiotherapy. To determine the D10 (the dose that reduces cell survival to 10%) of colorectal cancer cells to X-ray, we used a colony formation assay, and 6&#xa0;Gy was chosen for further experiments. Through TCGA, we found that miR-142-3p was upregulated in colorectal cancer cells. We then transfected miR-142-3p into colorectal cancer cells, constructing stable lines. We discovered that miR-142-3p promoted the radioresistance of colorectal cancer cells, and in vivo assays also demonstrated the same effect. Mechanistically, we identified FOXO4 as the direct target of miR-142-3p by Dual Luciferase Reporter Assay. RNA sequencing revealed that miR-142-3p enhanced DNA damage repair. Western blot analysis determined that the NHEJ pathway was involved in regulating this process, including DNA-PKcs, Ku80, Rad50, NBS1, MRE11, XRCC1, LIG3, et al. Together, our findings suggest that miR-142-3p plays a radioresistant role in colorectal cancer and reveals a potential therapeutic target to enhance the effectiveness of radiotherapy.</p> Clinical trial number <p>Not applicable.</p> Graphical Abstract <p></p>