Abstract： Objective： To investigate the mechanism by which Shiyan Prescription alleviates ulcerative colitis （UC）through the interleukin（IL）-23/IL-17 axis-mediated inhibition of T-helper 17（Th17）cell differentiation. Methods：A total of 48 mice were randomized into six groups（n=8）：blank，model，low-dose Shiyan Prescription， medium-dose Shiyan Prescription， high-dose Shiyan Prescription， and Mesalazine. Except for the blank group， all mice received 3% Dextran Sulfate Sodium（DSS）to establish the UC model. The low-， medium-， and high-dose Shiyan Prescription groups were administered 15 g（/ kg · d），30 g（/ kg · d）and 60 g（/ kg · d）Shiyan Prescription suspensions by gavage once daily. The Mesalazine group received 0.5 g（/ kg · d）Mesalazine suspension by gavage once daily. Disease Activity Index （DAI） was recorded daily. Colon length and intestinal mucosa inflammation scores （hematoxylin–eosin staining）were evaluated after the end of administration. Transcriptome analysis was performed to identify differentially expressed genes，followed by Gene Ontology（GO）function and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses. The qRT-PCR and western blot（WB）were employed to verify the expression of key genes and protein. Results：Compared with the blank group，the model group exhibited increased DAI scores （P＜0.05）. Compared with the model group， the groups of Shiyan Prescription（low， medium， and high-dose） exhibited reduced DAI scores and increased colon length（P＜0.05）. Hematoxylin-eosin staining revealed markedly reduced inflammation in the Shiyan Prescription groups，with the medium- and high-dose groups showing the greatest reductions. Transcriptomic analysis revealed significant enrichment of genes associated with the IL-23/IL-17 axis in the Th17 cell differentiation pathway， and both qRT-PCR and WB confirmed that Shiyan Prescription treatment downregulated the mRNA levels of IL-23， IL23R， STAT3， IL17A， and the protein levels of p-STAT3， RORγt， and IL-23， within this axis（P＜0.05）. Conclusion：Shiyan Prescription can alleviate intestinal inflammation in UC by inhibiting the IL-23/IL-17 signaling pathway，thereby inhibiting Th17 cell differentiation，restoring regulatory T cell （Treg）/Th17 balance，to re-establishing intestinal immune homeostasis and alleviate intestinal inflammation.