Chlorogenic acid (ChA)—one of the most abundant polyphenol compounds in the human diet—exerts anti-inflammatory activities. The aim of this study was to investigate the effect of ChA on gut microbiota in ulcerative colitis (UC). Colitis was induced by 2.5% dextran sulfate sodium (DSS) in C57BL/6 mice, which were on a control diet or diet with ChA (1 mM). The histopathological changes and inflammation were evaluated. Fecal samples were analyzed by 16S rRNA gene sequencing. ChA attenuated several effects of DSS-induced colitis, including weight loss, increased disease activity index, and improved mucosal damage. Moreover, ChA could significantly suppress the secretion of IFNγ, TNFα, and IL-6 and the colonic infiltration of F4/80⁺ macrophages, CD3⁺ T cells, and CD177⁺ neutrophils via inhibition of the active NF-κB signaling pathway. In addition, ChA decreased the proportion of Firmicutes and Bacteroidetes. ChA also enhanced a reduction in fecal microbiota diversity in DSS treated mice. Interestingly, ChA treatment markedly increased the proportion of the mucin-degrading bacterium Akkermansia in colitis mice. ChA acted as the intestine-modifying gut microbial community structure, resulting in a lower intestinal and systemic inflammation and also improving the course of the DSS-induced colitis, which is associated with a proportional increase in Akkermansia.