Abstract: Radiation-induced intestinal injury (RIII) is a common syndrome in the radiotherapy of abdominal and pelvic malignant tumors, heavily influencing the living quality, but no specific clinical regimens are available. Inulin is a naturally soluble dietary fiber. Clostridium butyricum (Cb) is a strict Gram-positive anaerobic bacillus, which can secrete and produce a large number of butyric acid, improving the intestinal barrier function and reducing the growth and colonization of opportunistic bacteria. A synbiotic of inulin gels (IG) and Cb exerts the synergistic effect of prebiotic intestinal retention and probiotics. In this study, an oral synbiotic of IG and Cb was applied for the prevention and treatment of RIII and the mechanisms were explored. Animal experiments were approved by the Ethics Committee of the Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (authorizing number: IACUC-DWZX-2022-525). An RIII mouse model was established after whole abdominal γ-ray radiation of 13 Gy. The synbiotic was intragastrically administered to the mice 1 h pre-radiation. Compared to the models, the mice of the synbiotic group had more regenerated intestinal crypts, longer villus and colon, and more tight junction protein on day 3.5. Moreover, the mice of the synbiotic group had an obvious increase in the relative abundance of gut microbiota on day 7, especially the amounts of multiple probiotics and short-chain fatty acid metabolites. On day 14, the mice of the synbiotic group had highly low permeability of intestinal mucosa according to the fluorescence labeling experiment, which was close to the normal level. Moreover, the mice of the synbiotic group showed a high decrease of proinflammatory factors including tumor necrosis factor-α and interleukin-6, close to the normal levels. Therefore, the oral synbiotic can alleviate the syndromes of RIII and improve the repair of damaged intestinal and colon tissues, and its protective effect is better than IG and Cb. The oral synbiotic is a safe and effective biological drug against RIII.