Radiotherapy serves as a crucial strategy in the treatment of colorectal cancer (CRC). However, its efficacy is often hindered by the challenge of radiation resistance. Although the literature suggests that some tRNA‐derived small RNAs (tsRNAs) are associated with various cancers, studies reporting the relationship of tsRNAs with cancer cell radiosensitivity have not been published yet. In our study, we utilized tsRNAs sequencing to predict differentially expressed tsRNAs in two CRC cells and their radioresistant cells, and 10 tsRNAs with significant differences in expression were validated by qPCR. The target genes of tRF‐16‐7X9PN5D were predicted and verified by the bioinformatics, dual‐luciferase reporter gene assay and western blotting analyses. Wound healing, colony formation, transwell invasion and CCK‐8 assays were performed to detect the effects of tRF‐16‐7X9PN5D on cell function and radiosensitivity. Western blotting evaluated the relationship between tRF‐16‐7X9PN5D and the MKNK‐eIF4E axis. Our findings demonstrated that tRF‐16‐7X9PN5D expression was substantially downregulated in radioresistant CRC cells. Furthermore, tRF‐16‐7X9PN5D could promote CRC cells' ability to proliferate, migrate, invade and obtain radiation resistance by targeting MKNK1. Finally, tRF‐16‐7X9PN5D could regulate eIF4E phosphorylation via MKNK1. This investigation indicated that tRF‐16‐7X9PN5D has an essential regulatory role in the radiation resistance of CRC by directly targeting MKNK1, and may be a new pathway for regulating the CRC radiosensitivity.