Abstract: Objective: To investigate the effect of Coix seed on colitis and intestinal flora in mice induced by dextran sulfate sodium (DSS) and explore its mechanism of action. Methods: UC was induced in mice by freely drinking 3.5% DSS solution for 7 days. The Coix seed extractions (CE) were given to the mice by gavage for intervention. The changes in body weight, disease activity index (DAI) scores, and colon length of mice were recorded. Colonic histopathological changes were observed by staining, levels of inflammatory factors in the colon of mice were detected, and 16S rDNA sequencing technology was used to analyze changes in fecal intestinal flora of mice. Pearson correlation coefficient was used to analyze the correlation between the relative abundance of key differential bacteria and the levels of inflammatory factors. Results: Compared with the model group, CE significantly improved weight loss, prevented the increase of DAI scores, restored the colon length (the colon length of CE group restored to 7.27 cm), decreased intestinal barrier damage and levels of inflammatory factors in mice (P<0.05), CE can also effectively regulate the changes in intestinal flora diversity in mice with UC. At the phylum level, CE reduced the relative abundance of Proteobacteria and increased the relative abundance of Bacteroidetes in colitis mice. At the genus level, CE increased the relative abundance of Akkermansia and Lactobacillus, which are associated with intestinal barrier repair. Further correlation analysis revealed a strong association between the relative abundance of key bacteria regulated by CE and the levels of inflammatory factors. Conclusion: Coix seed can alleviate colitis symptoms of mice in UC by regulating the intestinal flora, reducing levels of inflammatory factors, repairing colonic mucosal barrier, and thus relieving colonic inflammation.