The gut microbiota, including pathogenic microorganisms and probiotics, has been involved in tumor initiation and progression by regulating the components of intestinal flora. Canmei formula (CMF), a traditional Chinese medicine, chronicled in the Chuang Yang Jing Yan Quan Shu, has been clinically used as an adjuvant therapy to treat patients with colorectal carcinoma (CRC) in China. In this study, we investigate the treatment effect of CMF in the azoxymethane (AOM) and dextran sodium sulfate (DSS) induced and high-fat diet augmented colitis-associated colorectal cancer in vivo, and explore its mechanism of action. We found that CMF treatment relieved the inflammation and alteration of the gut microbiota and significantly inhibited the development of intestinal adenoma. Linear discriminant analysis showed that the flora diversity in the normal mice, model mice and CMF treatment mice was different. At the family level, the relative abundance of Desulfovibrionaceae decreased in CMF groups. The relative abundance of Desulfovibrionaceae were lower in the CMF groups than in model group, whereas Rikenellaceae and Alistipes were increased. Altogether our results indicate that CMF treatment ameliorate colitis-associated colorectal carcinogenesis by modulating the composition of the gut microbiota in vivo.