【Abstract】 Objective To optimize the method of combining azomethane oxide (AOM) and dextran sodium sulfate (DSS) to create a colitis-associated colon cancer (CAC) model, and to explore the pathogenesis of the intestinal flora in CAC. Method Model groups A and B were established by one and two injections of AOM, respectively, combined with free drinking of DSS, and a normal control group was injected intraperitoneally with normal saline combined with purified water (n=10 mice per group). The better modeling scheme was selected by comprehensive evaluation of the disease activity index score, colon length, tumor rate, and mortality. Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and tumor markers CA199, CEA, and CA724 were detected by enzyme-linked immunosorbent assay. Colon lesions were evaluated by hematoxylin and eosin (HE) staining. Changes in the intestinal microbiota in CAC mice were detected by 16S rDNA high-throughput gene sequencing analysis of mouse feces. Results Both single and enhanced AOM injections combined with DSS induced CAC mice; however, colon growths were larger, more closely arranged, and their morphological size was more consistent in group B compared with group A, with a tumor-formation rate of 100%. IL-6 and TNF-α levels were increased in the model group compared with the normal group (P<0.05, P>0.05, respectively). The CA199 and CEA levels were also significantly increased (P<0.05), but CA724 levels were not. Infiltration of inflammatory cells in the colon detected by HE pathology was accompanied by high-grade intraepithelial tumor-like changes on the surface of the lumen. The diversity and abundance of intestinal bacteria were decreased in CAC mice compared with normal mice: phyla Verrucomicrobiota and Actinobacteriota were significantly increased (P<0.05), Bacteroidota and Campilobacterota were significantly decreased (P<0.05). Akkermansia, Prevotellaceae, Ruminococcus, and Bifidobacterium were significantly increased (P<0.05), and Roseburia, Rikenellaceae_RC9_gut_group, Anaeroplasma, and Muribaculaceae were significantly decreased (P<0.05). Conclusion Two injections of AOM combined with 1.5% (1.5 g/100 ml) DSS induced CAC model mice with a high colon-tumorigenesis rate, uniform tumor morphology, and low mortality, and may thus be the preferred modeling scheme for pharmacodynamic experiments. Disorders or dysfunction of the intestinal flora may lead to increased permeability, loss of intestinal mucosal barrier function, and the release of enterogenic endotoxins, resulting in a sustained inflammatory response, as an indirect or direct cause of CAC pathogenesis.