ETHNOPHARMACOLOGICAL RELEVANCE: SiniSan (SNS), a traditional formula from the Treatise on Typhoid Fever, has been shown in modern clinical practice to effectively treat both ulcerative colitis (UC) and depression, although the underlying mechanisms remain incompletely understood. AIM OF THE STUDY: This study employed UPLC-Q-Orbitrap-MS, network pharmacology, molecular docking, and experimental validation to investigate SNS's mechanism in treating UC with comorbid depression. MATERIALS AND METHODS: UPLC-Q-Orbitrap-MS, in conjunction with network pharmacology and molecular docking, identified active constituents and potential targets of SNS. A UC model induced by a 3 % dextran sulfate sodium (DSS) solution was used for experimental validation. Therapeutic efficacy was assessed through behavioral tests, ELISA, routine blood tests, histopathology, immunofluorescence, Western blot, and qRT-PCR. RESULTS: SNS alleviated weight loss and inflammation in DSS-induced colitis in mice while exhibiting antidepressant effects in the open field test, forced swim test, and tail suspension test. Furthermore, SNS improved intestinal mucosal barrier function and restored hippocampal blood-brain barrier integrity. It inhibited microglial proliferation and neuroinflammation in the Hippocampus Cornu Ammonis 1 and dentate gyrus regions. Mechanistic analysis revealed that SNS mediates its effects on UC by modulating targets in the PI3K/AKT signaling pathway. CONCLUSIONS: SNS ameliorates UC with comorbid depression by restoring the integrity of both the intestinal mucosal barrier and the blood-brain barrier, alleviating DSS-induced colitis and neuroinflammation.