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Background Ulcerative colitis (UC) is a chronic inflammatory disease characterized by impaired mucosal homeostasis. Enteric glial cells (EGCs), a key component of the enteric nervous system, are activated during UC and overexpress S100 calcium-binding protein B (S100B). However, the underlying mechanisms remain unclear. This study aimed to elucidate the causes of EGC activation in colitis and to identify novel therapeutic strategies.Methods Clinical specimens and data were analyzed to assess gut microbiota alterations and bacterial invasion. Single-cell transcriptomics compared EGCs between UC patients and healthy controls. Fecal microbiota transplantation (FMT) was used to validate the effects of the microbiota and inflammation on EGCs. Flow cytometry was used to evaluate EGC activation and S100B expression in mouse models. Immunoprecipitation–mass spectrometry and co-immunoprecipitation identified S100B-interacting proteins. In vitro and in vivo assays examined the impact of S100B on macrophages and tested the effect of receptor for advanced glycation end products (RAGE) inhibition.Results Patients with UC exhibited gut microbiota dysbiosis and increased bacterial translocation across the intestinal epithelium. EGCs were activated with elevated S100B expression in both UC patients and colitis mice. FMT from UC patients increased the proportion of S100B + EGCs in the colon of recipient mice. EGC-derived S100B bound to RAGE on macrophages, thereby activating proinflammatory signaling pathways and further amplifying inflammation. Mechanistically, AHR functioned as an adaptor to bridge S100B and the CUL4B-RBX1 E3 ligase complex, facilitating S100B degradation via the ubiquitin-proteasome pathway, with K48-linked ubiquitination occurring at lysine 34. AHR deficiency reduced S100B ubiquitination and increased the abundance of S100B+ EGCs as well as S100B protein levels in the colon. Notably, supplementation with AhR ligands or RAGE inhibitors suppressed the S100B-RAGE axis, thereby alleviating colitis and enhancing the therapeutic efficacy of biologics.Conclusions These findings define a microbiota-EGC-macrophage axis in UC, in which AHR-dependent ubiquitination restrains S100B-driven inflammation. Targeting the S100B-RAGE pathway represents a promising strategy to enhance current therapies.