Ulcerative colitis (UC) is a major inflammatory disease worldwide. We previously demonstrated that licorice residue flavones (LFs) showed satisfactory efficacy in the treatment of UC. Therefore, research into the ingredients of LFs may lead to the discovery of novel anti-UC targets. In the current study, we separated licoflavone B (LB) from LFs and administered it to dextran sodium sulfate (DSS)-exposed C57BL/6 mice for 14 days. Our results demonstrated that high dose LB (120 mg/kg) significantly prevented DSS-induced weight loss, disease activity index (DAI) increase, histological damage, and colonic inflammation, indicating that LB has ameliorative effects on UC. We also investigated the composition of the intestinal barrier and microflora in an attempt to explore the mechanisms of LB against UC. As a result, we found that LB preserved the integrity of the colonic barrier by inhibiting colonic cell apoptosis and protecting the expression of occludin, claudin-1, and ZO-1. Moreover, LB reshaped the microflora composition by suppressing harmful bacteria (Enterococcus et al.) and boosting beneficial microorganisms (Bacteroides et al.). Further molecular exploration implied that LB exerted anti-UC activity through blocking the MAPK pathway. Here, we explored anti-UC activity of LB for the first time and clarified its mechanisms. These results will provide valuable clues for the discovery of novel anti-UC agents.