["Journal of Cellular Physiology, Volume 241, Issue 5, May 2026. ", "A probiotic formulation of Limosilactobacillus reuteri and Clostridium butyricum provides prophylactic protection against 5FU‐induced mucositis by strengthening the intestinal barrier, reshaping the microbiota to reduce pathogenic load, and regulating immune‐inflammatory response via TLR4‐mediated pathway.\n\n\n\n\n\n\nABSTRACT\n5‐fluorouracil (5FU)‐induced intestinal mucositis significantly limits its clinical utility and compromises patient outcomes. Building on our previous finding that a specific probiotic formulation, consisting of Limosilactobacillus reuteri BCRC 80379 and Clostridium butyricum MIYAIRI 588 (collectively referred to as LCs), mitigated cisplatin‐induced mucositis, this study evaluated the prophylactic efficacy and underlying mechanisms of LCs against 5FU‐induced mucositis in Wistar rats. Rats were divided into control, 5FU‐only, and 5FU+LCs groups. LCs was orally administered daily for 21 days before and throughout the 5FU treatment (50 mg/kg/day for 5 days). Intestinal tissues were analyzed using histological, molecular, and microbiological approaches. Pretreatment with LCs significantly attenuated 5FU‐induced small intestinal damage, as evidenced by improved mucosal structure (e.g., increased villus height, crypt depth, and goblet cell density), along with reduced inflammatory cell infiltration. Moreover, LCs reinforced the intestinal barrier by upregulating genes associated with the mucus layer (Muc2) and tight junctions (Ocln, Tjp1, and Cldn1). LCs also suppressed the TLR4/MyD88/NF‐κB pathway, leading to reduced levels of pro‐inflammatory mediators (e.g., TNF‐α, IL‐1β, and MPO) while elevating key regulatory mediators such as IL‐10 and secretory IgA (sIgA). Furthermore, microbial profiling revealed that LCs beneficially reshaped gut microbiota composition by reducing opportunistic pathogens (e.g., Escherichia‐Shigella and Enterobacter). These microbial changes were accompanied by a favorable immune modulation, including decreased pro‐inflammatory markers and increased homeostatic mediators. Collectively, these findings suggest that LCs mitigates 5FU‐induced mucositis through coordinated regulation of intestinal barrier integrity, immune responses, and gut microbiota, highlighting its potential as a probiotic‐based approach for managing chemotherapy‐induced intestinal injury."]