Increasing evidence shows that the gut microbiota contributes to the occurrence and development of colitis. Kudingcha (KDC), made from the leaves of Ilex kudingcha, could mitigate inflammation, however, little is known about the relationship between modulatory effect on gut microbiota by KDC and improvement of colitis. In this study, the attenuating effects of KDC extract (KDCE) on dextran sulfate sodium (DSS)-induced colitis and gut microbiota in C57BL/6 mice were investigated. It was found that the supplementation of KDCE could alleviate typical symptoms of IBD including weight loss, colon shortening, intestinal barrier damage, and decreases in the colitis disease activity index and pro-inflammatory cytokines. Moreover, KDCE supplementation could reverse the alteration of gut microbiota in the colitic mice by increasing the abundances of potential beneficial bacteria, e.g. Odoribacter, Prevotella and Helicobacter, and decreasing the abundances of potential harmful bacteria, e.g. Parabacteroides, Bacteroides, Turicibacter, Parasutterella and Lachnospiraceae. The levels of short-chain fatty acids in feces, cecum contents and serum were also regulated by KDCE. Furthermore, the correlation analysis suggested that KDCE could attenuate DSS-induced colitis which might be related to the alteration of gut microbiota. Therefore, the modulation of gut microbiota by KDCE might be a potential strategy for improving inflammation-driven diseases.