Based on serum metabolomics, the mechanism of action of Xianglian Pill in improving dextrane sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice was investigated. The main active ingredients of Xianglian Pill were detected using high-performance liquid chromatography (HPLC) technology. Male C57BL/6 mice were randomly divided into six groups: control, model, sulfasalazine (0.125 g/kg), low-dose Xianglian Pill (0.5 g/kg), medium-dose Xianglian Pill (1 g/kg), and high-dose Xianglian Pill (2 g/kg), with ten mice in each group. Except for the control group, all other groups were given 2.5% DSS to establish a ulcerative colitis (UC) model. During the modeling process, they were also administered orally for a total of 11 days. Record daily changes in mouse body weight, fecal characteristics, and occult blood status for disease activity index (DAI) scoring. The mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were detected using RT-qPCR; myeloperoxidase (MPO) activity was measured using a myeloperoxidase assay kit; histopathological changes in colon tissues were observed through HE staining; immunohistochemistry was used to detect the expression of zonula occludens-1 (ZO-1) and Occludin; non-targeted metabolomics was employed to analyze the metabolite levels in serum samples from UC mice; and Western blot was used to detect the expression of key proteins in major metabolic pathways. The results indicated that Xianglian Pill could significantly reduce the DAI score in UC mice, alleviate colonic tissue damage, decrease inflammatory cell infiltration, and downregulate the levels of pro-inflammatory factors (IL-6, IL-1β, and TNF-α), MPO activity, as well as the expression levels of ZO-1 and Occludin. Metabolomic analysis revealed a total of 38 potential differential metabolites associated with the intervention of Xianglian Pill in UC mice, primarily involving sphingolipid metabolism, glycerophospholipid metabolism, and linoleic acid metabolism. Sphingolipid metabolism is closely related to the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. Western blot results indicate that Xianglian Pill can significantly downregulate the protein expression of sphingosine kinase 1 (SPHK1) and phosphoglycerate dehydrogenase (PHGDH), thereby reducing the activation of the PI3K-AKT pathway. In summary, Xianglian Pill can significantly improve the disease state of DSS-induced UC mice, and its mechanism of action may be closely related to regulating the sphingolipid metabolism-PI3K-AKT pathway.