Abstract: Objective: To explore the effect of gardenia yellow pigment (GYP) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in rats. Methods: Four groups of rats were established: normal group, model group, positive control group, and GYP intervention group. The health status of the rats was recorded on the day of sampling, and the disease activity index (DAI) score was calculated. The weight change, DAI score, colon tissue morphology, short chain fatty acids (SCFAs) content and the composition of intestinal flora were compared between the four groups. Furthermore, preliminary analysis was conducted on the mechanism of GYP in alleviating UC in rats through network pharmacology and molecular docking. Results: The results indicated that GYP not only effectively alleviated the symptoms of ulcerative colitis such as body mass loss, hematochezia, diarrhea, and the shortening of colon length and colon tissue injury in rats. Moreover, GYP significantly increased the relative abundance of Firmicutes and the content of acetic acid (P<0.05), and significantly reduced the relative abundance of Bacteroidetes and Spirochetes (P<0.05). Through network pharmacology screening, crocetin was identified as a key component for alleviating UC in GYP, and 38 intersection targets and 86 KEGG pathways were identified. In addition, molecular docking results demonstrated that crocetin had good affinity for core targets for alleviating UC, among which ACE had the highest binding degree. Conclusion: GYP may alleviate intestinal damage and microbial disruption in DSS induced UC rats by increasing the content of short chain fatty acids and maintaining the homeostasis of gut microbiota.