Aflatoxin M₁ (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. The combined effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX cocultures. Our results showed that the mixtures of AFM1+OTA, AFM1+ZEA, and AFM1+ZEA+OTA significantly decreased transepithelial resistance values and increased the paracellular flux of lucifer yellow and FITC-dextrans on Caco-2/HT29-MTX cells, which indicated an increased epithelial permeability. Although the expression levels of tight junction (TJ) proteins did not change significantly, immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures, namely, claudin-3, claudin-4, occludin, and zonula occludens-1. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated with the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered.