〔Abstract〕 Objective To investigate the metabolic mechanisms of Limosilactobacillus reuteri (L.reuteri) in alleviating chemotherapy-induced intestinal mucosal injury (CIMI). Methods C57BL/6 mice were randomly divided into the Control group, Model group, and L. reuteri treatment group (n = 8 per group). Mice in the L. reuteri group were intragastrically administered L. reuteri (1 × 109 CFU/mL) for 10 consecutive days. From day 5, mice in the Model and L. reuteri groups were intraperitoneally injected with 5-FU (65 mg/kg) for 6 consecutive days to establish a CIMI model.Body weight changes, colon length, and related indicators were recorded. Intestinal histopathological injury was assessed by H&E staining with measurement of villus height and crypt depth. The expression levels of zonula occludens-1 (ZO-1), Occludin, Claudin-4, Muc1, and Muc2 in ileal tissues were determined by RT-qPCR and immunohistochemistry to evaluate intestinal barrier function. In addition, untargeted metabolomics was performed on cecal contents from each group to analyze metabolic profiles and enrichment of differential metabolic pathways. Results L.reuteri intervention significantly alleviated body weight loss (P<0.001) and colon shortening (P<0.05) in CIMI mice, effectively reducing ileal tissue pathological damage, and upregulating the expression of ZO-1, Occludin, Claudin4, Muc1, and Muc2 (P<0.05). Untargeted metabolomics analysis showed that L. reuteri treatment reversed the abnormal metabolic profile in the cecal contents of CIMI mice, bringing it closer to normal. Various differential metabolites, such as acylcarnitines and polyunsaturated fatty acids, might play an important role in L. reuteri-mediated alleviation of CIMI. These differential metabolites were primarily enriched in key pathways such as linoleic acid metabolism, tryptophan metabolism, and unsaturated fatty acid biosynthesis. Conclusion This study reveals that L. reuteri improves intestinal barrier function and alleviates CIMI by regulating the intestinal metabolic profile, particularly lipid and amino acid metabolism. It provides a novel potential strategy for the prevention and treatment ofCIMI.