RIO Kinase 1 (RIOK1) is involved in various pathologies, including cancer. However, the role of RIOK1 in radioresistance of colorectal cancer (CRC) remains largely unknown. In this study, we reported that RIOK1 was overexpressed in rectal cancer tissue with weaker tumor regression after neoadjuvant chemoradiotherapy (neoCRT). Moreover, higher RIOK1 expression predicted a poor prognosis in patients with rectal cancer. Blockade of RIOK1 using Toyocamycin, a pharmacological inhibitor of RIOK1, or by knocking down its expression, decreased the resistance of CRC cells to radiotherapy in vitro and in vivo. A mechanistic study revealed that RIOK1 regulates radioresistance by suppressing the p53 signaling pathway. Furthermore, we found that RIOK1 and Ras-GAP SH3 domain binding protein 2 (G3BP2) interact with each other. RIOK1 phosphorylates G3BP2 at Thr226, which increases the activity of G3BP2. RIOK1-mediated phosphorylation of G3BP2 facilitated ubiquitination of p53 by murine double minute 2 protein (MDM2). Altogether, our study revealed the clinical significance of RIOK1 in CRC, and therapies targeting RIOK1 might alleviate the CRC tumor burden in patients.