ETHNOPHARMACOLOGICAL RELEVANCE: Gegen Qinlian decoction (GQD) is a traditional Chinese medicine derived from Treatise on febrile diseases and is clinically used for the treatment of acute ulcerative colitis (UC). However, the potential mechanism of GQD treatment for UC remains elusive. AIM OF STUDY: In this study, we aimed to explore the involvement of gut microbiota-related tryptophan metabolism in mediating protective effects of GQD against intestinal barrier damage. MATERIALS AND METHODS: Mice with colitis were treated with 3% dextran sulfate sodium (DSS) for 6 days. The therapeutic effects of GQD in UC mice were examined based on body weight, disease activity index (DAI), organ index, length and pathological changes in the colon. The distribution of fluorescein isothiocyanate dextran (FITC-dextran) in the intestinal tract was observed using small animal imaging, while concentration of FITC-dextran in serum was detected using a fluorescein microplate analyser. Bacterial infiltration in colon tissues was observed by fluorescence in situ hybridisation (FISH), and the bacterial load in mesenteric lymph nodes (MLNs) was further examined through bacterial culture. Subsequently, colonic goblet cells were detected using Alcian blue staining. The tight junctions of the colonic epithelium were observed using transmission electron microscopy, and the expression of tight junction proteins was detected by immunofluorescence (IF) and western blot. In addition, flow cytometry was used to analyse the proportion of interleukin-22-positive (IL-22) ILC3 cells in lamina propria lymphocytes, and the content of IL-22 in colon homogenates was determined using an ELISA kit. In addition, targeted tryptophan metabolomics was used to detect the concentration of indole derivatives produced by tryptophan metabolism in faeces, and 16S rDNA was used to investigate the composition and abundance of gut microbiota-related tryptophan metabolism. RESULTS: Administration of GQD significantly alleviated the pathological symptoms, including weight loss, increased DAI score, changes in organ index, colon shortening, and colon pathological injury in UC mice. In addition, GQD reduced the diffusion of FITC-dextran in the intestinal tract, the content of FITC-dextran in serum, and bacterial infiltration in MLNs and colon tissues. Additionally, GQD significantly increased the number of colonic goblet cells, repaired the structure of epithelial tight junctions and increased the expression of tight junction proteins. Furthermore, GQD significantly increased the proportion of IL-22 ILC3 in the lamina propria, the expression of CYP1A1 protein in colon tissue, and the level of IL-22 in colon homogenates. However, the above protective effects of GQD were inhibited by co-administration of GQD and aryl hydrocarbon receptor (AhR) antagonist. Additionally, GQD restored the content of indole derivatives generated by tryptophan metabolism, regulated the diversity of the gut microbiota, and significantly increased the abundance of genes related to tryptophan metabolism. CONCLUSION: Our results confirmed that GQD repaired the damaged intestinal barrier in UC mice by regulating gut microbiota-related tryptophan metabolism and restoring the generation of indole derivatives to activate AhR-mediated IL-22 production.