Background: Prunetin (Pru), a bioactive flavonoid present in Caulis spatholobi, has been reported to possess a variety of pharmacological effects. However, the therapeutic effect and underlying mechanisms of Pru in inflammatory bowel disease have not been previously investigated. This study aimed to explore the protective action of Pru on dextran sulfate sodium (DSS)-induced colitis by network pharmacology and experimental validation. Methods: The corresponding genes of Pru were predicted using the Comparative Toxicogenomics Database (CTD) and SwissTargetPrediction. Differentially expressed genes (DEGs) between the control rats and DSS-induced colitis rats were identified from the microarray profile GSE54005. Cytoscape software was used to visualize the protein-protein interaction (PPI) networks of overlapped targets. Besides, an animal experiment was performed to verify the therapeutic effect of Pru on colitis. Results: 21 potential targets related to colitis treated by Pru were identified. The topological analysis revealed that Tnf, Il6, and Il1b were the key genes. The KEGG and GO enrichment analyses showed that key targets were enriched in the inflammatory bowel disease, IL-17 signaling pathway, TNF signaling pathway, regulation of inflammatory response, and acute inflammatory response. The in vivo experiment revealed that Pru improves pathological injury and alleviated colitis symptoms via the regulation of Tnf, Il6, and Il1b, which were involved in the acute inflammatory response. Conclusion: Our findings demonstrated that Pru alleviates colitis symptoms through the regulation of inflammatory response, which provides a scientific basis for Pru in preventing and treating colitis.