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Growing evidence suggests that gut microbiota are associated with atherosclerosis (AS). However, the functional heterogeneity of each gut segment gives rise to regional differences in gut microbiota. We established a rat model of AS by feeding the rats a high-fat diet for a long period. The pathological and microbiota changes in the ileum and colon of the rats were examined, and correlations between AS and microbiota were analyzed. The aortic mesothelium of the experimental rats was damaged. The intima showed evident calcium salt deposition, indicating that the AS rat model was successfully developed. We noted varying degrees of pathological damage in the ileum and colon of the experimental rats. The 16S rDNA high-throughput sequencing showed significant differences in α-diversity, β-diversity, and microbiota comparisons in the ileum and colon. Furthermore, the ileum and colon of AS rats showed varying degrees of intestinal microbiota disturbance. This article contributes to the study of the relationship between the microbiota in different regions of the gut and AS, and provides new approaches in gut microbiota intervention for the treatment of AS.