Ulcerative colitis (UC), a chronic inflammatory bowel disease with increasing global incidence and limited therapeutic options, underscores the urgent need for novel multi-target agents. Swertiamarin (STM), a secoiridoid glycoside derived from traditional medicine, exhibits anti-inflammatory properties, but its pharmacological mechanisms in UC remain unclear. In this study, we integrated network pharmacology and experimental validation to systematically decipher STM's therapeutic effects. Network analysis identified 67 overlapping targets between STM and UC, which were significantly enriched in key pathways such as Toll-like receptor 4/Nuclear factor-kappa B (TLR4/NF-κB), Interleukin-17 (IL-17), and apoptosis. Molecular docking and protein-protein interaction (PPI) networks prioritized core targets such as TLR4, Caspase-3 (CASP3), and Prostaglandin-endoperoxide synthase (PTGS2). In a dextran sulfate sodium (DSS)-induced murine UC model, STM treatment significantly alleviated colitis severity, evidenced by reduced disease activity index (DAI), attenuated colon shortening (56.5% improvement vs. DSS group, p < 0.01), and restored histological integrity. Mechanistically, STM suppressed TLR4/NF-κB signaling, decreasing phosphorylated Inhibitor of NF-κB alpha (p-IκBα) (1.84 ± 0.33 vs. DSS 2.32 ± 0.28) and NF-κB (1.62 ± 0.39 vs. DSS 2.33 ± 0.38), while downregulating pro-inflammatory mediators (TNF-α, Interleukin-1β) and elevating anti-inflammatory Interleukin10 (IL-10) (98.33 ± 4.13 vs. DSS 61.70 ± 6.70, p < 0.01). Furthermore, STM reduced intestinal epithelial apoptosis (20 ± 2 vs. DSS 55 ± 3, p < 0.01) and modulated systemic immune responses by normalizing lymphocyte/neutrophil ratios. These findings reveal STM's multi-target efficacy in UC, bridging traditional medicine with modern mechanistic insights, and position it as a promising candidate for further clinical development.