Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD). Scutellaria baicalensis Georgi (SBG), a widely used traditional Chinese medicine, exhibits anti-inflammatory and antioxidant properties. Scutellaria Baicalensis stem-leaf total flavonoids (SSTF) ameliorate colonic pathological damage in UC mouse model. However, its mechanism of action remains to be further explored. Aim of study: To investigate the therapeutic effects of SSTF on UC mice and the underlying mechanisms. Materials and methods: Construct a mouse model of UC and evaluate therapeutic efficacy by assessing clinical indicators and histopathological changes. Integrated gut microbiota and proteomics identified core targets associated with SSTF, and target validation was performed by immunohistochemistry. Results: Our results revealed that SSTF significantly improves clinical indicators (colon length, body weight, DAI score, spleen index, TNF-α, IL-10) in UC mice. Pathological damage was reduced, intestinal mucosal barrier impairment was improved, and core proteins (MUC2, ZO-1) were elevated. Proteomics identified 1116 differentially expressed genes. SSTF significantly regulated the intestinal flora structure of UC mice to promote the proliferation of beneficial bacterial genera. Three core targets (NR1D1, AMBRA1, YAP1) were validated by immunohistochemistry, confirming their roles in the treatment of UC with SSTF. Conclusion: SSTF improves UC and attenuates histopathological damage. These findings further elucidate the treatment mechanism of SSTF and provide new ideas for the treatment of UC.