Berberine (BBR), a major active constituent of Rhizoma coptidis, was reported to exert beneficial effects on intestinal mucositis (IM) induced by 5‐fluorouracil (5‐FU). However, the bioavailability of BBR is extremely low, and its metabolites were perceived to contribute to its prominent pharmacological activities. Oxyberberine (OBB) is a gut metabolite of BBR, which has been reported to have a superior anti‐inflammatory effect in experimental colitis. However, its anti‐inflammatory effects against 5‐FU‐induced IM mice have not yet been investigated. Hence, the purpose of this study was to reveal the protective effects of OBB on IM induced by 5‐FU and investigate its potential underlying mechanism. The IM mice model was induced by receiving 5‐FU (60 mg/kg, i.p.) for five days. Meanwhile, BBR (50 mg/kg) and OBB (12.5, 25, and 50 mg/kg) were given prior to 30 min intraperitoneal injection of 5‐FU for seven days. Results indicated that OBB ameliorated body weight loss, anorexia, diarrhea, and histopathological damage in 5‐FU‐induced IM mice. After OBB administration, the amounts of MDA, SOD, and GSH altered by IM were remarkably restored. OBB was also observed to dramatically decrease the levels of TNF‐α, IL‐8, IL‐6, COX‐2, and iNOS and promote the release of IL‐10. Besides, OBB distinctly upregulated the mRNA expressions of PCNA, ZO‐1, occludin, and mucin‐1, which could improve intestinal homeostasis in IM mice. OBB also blocked the activation of the upstream TLR4/MyD88 signaling pathway, and then it inhibited the phosphorylation of the NF‐κB and MAPK pathways. Importantly, compared with BBR, OBB displayed a superior therapeutic effect to BBR in alleviating 5‐FU‐induced IM mice. These results indicated that OBB has considerable potential to become a novel candidate drug against IM.