Rhoifolin (ROF), a flavonoid compound isolated from citrus plants, has been shown in modern research to possess a range of important biological activities, including anti-inflammatory and anti-tumor properties.In this study, we induced ulcerative colitis (UC) in mice using dextran sulfate sodium (DSS) and treated them with ROF during the therapeutic phase.The results showed that ROF significantly alleviated the weight loss, colon shortening, and histopathological damage in the colon tissues of the mice, while also improving intestinal barrier function by restoring the expression of ZO-1 and E-cadherin. 16S rDNA sequencing analysis indicated that ROF treatment significantly altered the diversity and composition of the gut microbiota, increasing the relative abundance of Bacteroidetes, and Lactobacillus. Flow cytometry analysis revealed that ROF significantly reduced the proportion of Th17 cells in peripheral blood while increasing the proportion of Treg cells. Molecular docking analysis demonstrated that ROF could effectively bind to the Th17 cell transcription factor RORγt and the Treg cell transcription factor FOXP3, suggesting a potential regulatory effect. Further transcriptomic analysis revealed that ROF downregulated the expression of genes associated with the IL-17 signaling pathway, including IL-17A, TNF-α, NF-κB, CXCL10, and CXCL5, further supporting its anti-inflammatory mechanism by inhibiting the IL-17 pathway.In conclusion, we provides the first evidence that ROF alleviates DSS-induced colonic inflammation by modulating gut microbiota diversity, restoring the Th17/Treg cell balance, and inhibiting the IL-17 signaling pathway.