The ameliorative effects of carnosic acid (CA) on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice were assessed. Ulcerative colitis was induced by the oral administration of 3% DSS via distilled drinking water for seven days. A total of 60 mice were randomly divided into five groups: blank control (CK), DSS model (DSS), low-dose carnosic acid (CAL), high-dose carnosic acid (CAH), and mesalazine (PC). The ameliorative effects of CA were evaluated based on body weight, disease activity index (DAI) score, colonic histopathology, and changes in intestinal permeability. To investigate the possible mechanism of CA, the activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), the level of malondialdehyde (MDA), the expression level of tight junction proteins, including ZO-1 and occludin, and the changes in intestinal flora in mice were examined. When the CA and DSS groups were compared, CA intervention was found to reduce weight loss and the DAI score and ameliorate the pathological damage in colonic tissues in UC mice. The MPO activity was also found to significantly decrease in the CA groups. The MDA content in the colon tissue was reduced by 13.75% and 70%, respectively (P<0.05), while the SOD activity increased by 6.12- and 9.62-fold, respectively (P<0.05), in the CAL and CAH groups. Notably, the intestinal permeability was significantly reduced, and the expression levels of ZO-1 and occludin were restored. Gavage of 50 mg/kg CA enhanced the abundance ratios of Firmicutes and Bacteroides and restored the decrease in the abundance of beneficial bacteria, such as Akkermansia, caused by DSS. The relative abundance of detrimental bacteria, such as Alistipes, was also reduced. Overall, CA may mitigate UC by lowering the levels of oxidative stress, protecting the intestinal barrier, and regulating the composition of the intestinal microbial community.