2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most abundant heterocyclic amines, is a common carcinogen produced in thermally processed protein-rich foods. Studies have demonstrated that PhIP could induce colon tumors in rodents, leaving mechanisms uncovered. This study aims to investigate the mechanism of PhIP-induced colon injury in a rat model. The results of 16S rRNA gene sequencing and metabolomics showed that PhIP disrupted intestinal bacterial composition and affected the glycerophospholipid metabolism and linoleic acid metabolism. Simultaneously, the lipid metabolism function in the intestinal flora was inhibited by PhIP. Notably, transcriptomics revealed that PhIP remarkably inhibited the expression of gene sets associated with steroid hormone biosynthesis, fatty acid elongation, fatty acid degradation, and glycerolipid metabolism pathways in the colon. The results provide new perspectives to study the mechanism of PhIP-induced colon injury and theoretical bases for further understanding the toxicity of PhIP.