Background <p>Advances in medical science improved cancer treatment outcomes, and radiotherapy became a common modality used by 50% of cancer patients. However, radiotherapy induces gastrointestinal distress, and it can severely impair patients’ quality of life. During irradiation, the gut microbiome is critical in maintaining intestinal health and influences the body’s response. This study examines the alterations in the gut microbiome and serum metabolites of prostate cancer patients undergoing radiotherapy. We investigate the potential radioprotective effects of <i>Alistipes (</i>A.<i>) onderdonkii</i>.</p> Methods <p>Ten patients with prostate cancer receiving radiotherapy were included in this study. Fecal and serum samples were collected, and diarrhea symptoms were monitored along with each radiotherapy section.</p> Results <p>After radiotherapy, nine microbiomes and 129 serum metabolites displayed significant changes associated with irradiation-induced gastrointestinal toxicity. Dehydroascorbic acid and <i>A. onderdonkii</i> found a correlation in the analysis between metabolites and the microbiome. Administration of <i>A. onderdonkii</i> significantly improved survival and reduced intestinal damage in mice after radiation exposure, suggesting <i>A. onderdonkii</i> as a protective agent in radiotherapy.</p> Conclusions <p>This result highlights <i>A. onderdonkii</i> as a potential microbial candidate for mitigating irradiation-induced damage in the gastrointestinal tract. It appears to alleviate oxidative stress and support mucosal integrity, thereby promoting resistance to radiation-induced injury.</p> Impact <p>This finding establishes the foundation for developing microbiome-based therapeutics for the gut health of cancer patients undergoing radiotherapy.</p>