BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by persistent inflammation of the colon. Glycyrrhiza uralensis extract (ULE), a traditional medicinal plant extract, is globally recognized for its anti-inflammatory and gastrointestinal therapeutic effects. However, the precise mechanisms underlying the therapeutic benefits of ULE in UC management remain poorly understood. PURPOSE: This study aimed to elucidate the pharmacological effects of ULE and its primary active constituent, 18beta-glycyrrhetinic acid (18beta-GA), on the colonic epithelium in UC rat models. STUDY DESIGN AND METHODS: We employed a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC rat model and a TNBS-LPS-induced UC Caco-2 cell model to explore the protective mechanisms of ULE and 18beta-GA. The methodologies included UPLC-Q/TOF-MS chemical analysis in chemico, network pharmacological screening in silico, histopathological and flow cytometry evaluations in vivo, and molecular biological validation in vitro. RESULTS: ULE significantly alleviated UC symptoms in rats, including weight loss, diarrhea, and rectal bleeding, particularly at a dosage of 100 mg/kg. Network pharmacology and transcriptomic analyses identified the Wnt pathway as a pivotal hub linking inflammation, epithelial proliferation, and tight junction integrity. ULE treatment reduced apoptosis (from 13.24% to 8.34%), CD3(+) T cells (from 13% to 6.04%), and CD45RA(+) B cells (from 7.23% to 1.67%) in colonic tissues. Furthermore, ULE restored tight junction protein expression, notably increasing Occludin (2.5x) and ZO-1 (5.3x). Similarly, 18beta-GA activated the Wnt/beta-catenin signaling pathway and elevated tight junction protein expression, thereby protecting against UC-like damage in the Caco-2 cell model. These protective effects were potentiated by the Wnt pathway agonist SKL2001 and diminished by the antagonist IWR-1. CONCLUSION: ULE and its active constituent, 18beta-GA, exhibit protective effects against TNBS-induced colonic epithelial injury and tight junction protein disruption by upregulating the Wnt/beta-catenin signaling pathway, making them promising therapeutic candidates for UC management.