Object This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells. Methods: Microarray analysis was used to screen out IncRNAs differentially expressed in radio-resistant CRC cell lines. Expression levels of OIP5-AS1, miR-369-3p and DYRKIA in CRC cell lines were measured by qRT-PCR. Protein expression of DYRKIA was determined by western blot. The target relationships among OIP5-AS1, miR-369-3p and DYRKIA were validated by dual luciferase reporter assay. Impacts of OIP5-AS1 or DYRKIA on CRC cellular activity and apoptosis were investigated by MIT assay, clonogenic survival assay and flow cytometry to analyze OIP5-AS1 or DYRK1A's effect on radioresistance of CRC cells. Results: LncRNA OIP5-AS1 and DYRKIA were down-regulated in radio-resistant CRC cell lines. OIP5-AS1 suppressed the expression of miR-369-3p, thus up-regulating DYRKIA, the downstream gene of miR-369-3p. OIP5AS1 and DYRKIA impaired cell clonogenic survival and promoted cell apoptosis after irradiation, improving radiosensitivity of CRC cells. Conclusion: LncRNA OIP5-AS1 suppressed cell viability, promoted radio-induced apoptosis, and enhanced the radiosensitivity of CRC cells by regulating DYRKIA expression through miR-369-3p.