Objective: The Zuo Jin pill (ZJP), a classic formula for treating gastrointestinal diseases, has demonstrated good efficacy in the treatment of ulcerative colitis (UC). In this study, we aimed to elucidate the mechanism of action of ZJP in UC through network pharmacology and experimental validation. Methods: Bioactive compounds and targets of ZJP, along with UC-related targets, were retrieved from public databases. The intersecting targets of ZJP and UC were visualized using a Venn diagram. Protein–protein interactions and complex target networks were established using Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to enrich the biological functions and pathways of potential targets and to establish a biological process-target-pathway network. Relevant pathways were screened based on literature review and node size, and the interactions between ZJP and UC as well as hub target proteins were verified through animal experiments. Results: The findings suggested that STAT3, AKT1, EGFR, PIK3R1, and various cancer pathways exhibited potential as pivotal targets and biological processes contributing to the therapeutic efficacy of ZJP in UC. These targets were intricately associated with molecular functions, drug response, protein autophosphorylation, and protein kinase binding. Subsequent experimental intervention using ZJP on dextran sulfate sodium-induced UC mice revealed that its mode of action may involve inhibiting the PI3K/AKT/mTOR signaling pathway, mitigating intestinal mucosal inflammation, and modulating apoptosis in intestinal epithelial cells. Conclusions: The findings suggested that STAT3, AKT1, EGFR, PIK3R1, and various cancer pathways exhibited potential as pivotal targets and biological processes contributing to the therapeutic efficacy of ZJP in UC. These targets were associated with drug response, protein autophosphorylation, and protein kinase binding. Further experiments revealed that its mode of action may involve inhibiting the PI3K/AKT/mTOR signaling pathway, mitigating intestinal mucosal inflammation, and modulating apoptosis in intestinal epithelial cells.