ETHNOPHARMACOLOGICAL RELEVANCE: Buzhongyiqi Granules (BZYQ), a classical herbal formulation derived from Pi Wei Lun, is traditionally used to regulate Qi and blood circulation while enhancing immune function, particularly for gastrointestinal disorders. However, its protective effects and mechanisms on ulcerative colitis (UC) remain unclear. AIM OF THE STUDY: This study aimed to identify the blood-entering bioactive components of BZYQ and elucidate its immune-modulatory mechanisms in UC using an integrated strategy combining medicinal chemistry, bioinformatics analysis, molecular docking, and experimental validation. MATERIALS AND METHODS: The chemical profile and serum pharmacochemistry components (SPCs) of BZYQ were identified via UPLC-QE-Orbitrap mass spectrometry. Network pharmacology and molecular docking predicted UC-related targets and pathways. In vivo, experimental colitis was induced via 3 % dextran sulfate sodium solution administration for 7 days, followed by 10-day BZYQ treatment. Anti-inflammatory effects were validated through flow cytometry, qPCR, Western blotting, and in vitro bone marrow-derived macrophage (BMDM) cultures. RESULTS: Fifty-four compounds were identified in BZYQ, with seven SPCs (astragaloside IV, licoflavone A, 18beta-glycyrrhizinic acid, glabrolide, ganoderic acid X, complanatuside, and isosakuranin) directly related to UC pathogenesis. Molecular docking confirmed high-affinity interactions between glabrolide and IL-6. BZYQ alleviated colitis by suppressing IL-6/STAT3 axis, reprogramming macrophage polarization, increasing regulatory T cell frequency, and suppressing naive CD4(+) T cell differentiation. CONCLUSIONS: This study is the first to define BZYQ's component basis and cellular immunological mechanisms in UC alleviation. These findings contribute to the development and application of traditional Chinese medicine prescriptions.