We previously found greater reduction of colon cancer (CC) biomarkers for red wheat compared to white wheat regardless of refinement state. In the present study we examined whether the phenolic-rich aleurone and testa layers are drivers of chemoprevention by red wheat and their influence on gut microbiota composition using a 1,2-dimethylhydrazine-induced CC rat model. Rats were fed a low-fat diet (16% of energy as fat), high-fat diet (50% of energy as fat), or high-fat diet containing whole red wheat, refined red wheat, refined white wheat, or aleurone- or testa-enriched fractions for 12 weeks. Morphological markers (aberrant crypt foci, ACF) were assessed after methylene blue staining and biochemical markers (3-nitrotyrosine [3-NT], Dclk1) by immunohistochemical determination of staining positivity within aberrant crypts. Gut microbiota composition was evaluated from 16S rRNA gene sequencing of DNA extracted from cecal contents. Relative to the high-fat diet, the whole and refined red wheat, refined white wheat, and testa-enriched fraction decreased ACF, while only the refined red wheat and aleurone-enriched fraction decreased 3-NT. No significant differences were observed for Dclk1. An increase in microbial diversity was observed for the aleurone-enriched fraction (ACE index) and whole red wheat (Inverse Simpson Index). The diet groups significantly modified overall microbiome composition, including altered abundances of Lactobacillus, Mucispirillum, Phascolarctobacterium, and Blautia coccoides. These results suggest that red wheat may reduce CC risk through modifications to the gut microbiota and nitrosative stress, which may be due, in part, to the influence of dietary fiber and the phenolic-rich aleurone layer.