Aflatoxin M₁ (AFM₁) is known as one of the hydroxylated metabolites of aflatoxin B₁ (AFB₁) and the most threatening aspect of AFB₁ contamination. It could lead to hepatotoxicity and hepato-carcinogenicity, and possess intestinal cytotoxicity. However, little was known about the potential mechanisms of the intestinal cytotoxic effect. The aim of this study was to investigate the intestinal barrier damage induced by AFM1 via transcriptome analysis. Gene expression profiling was analyzed to comparatively characterize the differentially expressed genes (DEG) after differentiated Caco-2 cells were exposed to different concentrations of AFM₁ for 48 h. A total of 165 DEGs were significantly clustered into 2 downregulated patterns. The Search Tool for Retrieval of Interacting Genes (STRING) based protein-protein interaction (PPI) network analysis suggested 23 key enzymes mainly participate in regulation of cell cycle. Q-PCR analysis was performed to validate that the 12 key genes BUB1, BUB1B, MAD2L1, CCNA2, RB1, CDK1, ANAPC4, ATM, KITLG, PRKAA2, SIRT1 and SOS1 were involved. This study is the first to our knowledge to reveal that the toxicity of AFM1 against intestinal function might be at least partly due to the occurrence of cell cycle arrest.