Objective The aim of this study is to establish a rat model of inflammation-cancer transformation of inflammatory bowel disease (IBD) and explore its intestinal flora characteristics. Methods Adult male Wistar rats were randomly divided into control group and model groups (M1, M2, M3) with different DSS intervention cycles. Rats in the model group were all induced colitis-cancer transformation by single intraperitoneal injection of AOM combined with free drinking of DSS in different cycles, during which DAI scores were recorded. Rats in M1, M2 and M3 groups were killed at the end of the first, second and third cycles of DSS, and spleen, colon tissue and colon contents were collected. Hematoxylin and eosin (H E) staining and transmission electron microscope (TEM) were used to evaluate the histological damage and carcinogenesis of colon in each group, and the characteristic changes of intestinal flora were analyzed by 16S rRNA sequencing technology. Results AOM/DSS resulted in significant increase of DAI score, shortening of colon and increase of spleen index. From M1 to M3 group, the intestinal mucosal barrier was gradually destroyed, and the pathological score was gradually increased. Abnormal crypt focus, polyp, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and mucosal carcinoma appeared in turn. At the same time, the pathological evolution process shows similar characteristics to the carcinogenesis of human inflammatory bowel disease. In 16S rRNA sequencing, two differential abundance testing tools, Wilcoxon and ALDEx2, were used to screen out that the changes of flora abundance represented by Bacteroidetes and Monoglobus may be involved in the progress of colitis-cancer transformation, and the functions of differential flora were mainly enriched in metabolic pathways such as lipid metabolism and carbohydrate metabolism. Conclusions The rat model induced by AOM/DSS can dynamically simulate the pathological characteristics of colitis-cancer transformation, accompanied by changes in the abundance of specific intestinal flora, which may be closely related to the metabolic pathway mediated by flora.