Ochratoxin A (OTA), an important mycotoxin that occurs in food and animal feed, has aroused widespread concern in recent years. Previous studies have indicated that OTA causes nephrotoxicity, hepatotoxicity, genotoxicity, immunotoxicity, cytotoxicity, and neurotoxicity. The intestinal toxicity of OTA has gradually become a focus of research, but the mechanisms underlying this toxicity have not been described. Here, differentiated Caco-2 cells were incubated for 48 h with different concentrations of OTA and transcriptome analysis was used to estimate damage to the intestinal barrier. Gene expression profiling was used to compare the characteristics of differentially expressed genes (DEGs). There were altogether 10,090 DEGs, mainly clustered into two downregulation patterns. The Search Tool for Retrieval of Interacting Genes (STRING), which was used to analyze the protein–protein interaction network, indicated that 24 key enzymes were mostly responsible for regulating cell apoptosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was used to validate eight genes, three of which were key genes (CASP3, CDC25B, and EGR1). The results indicated that OTA dose-dependently induces apoptosis in differentiated Caco-2 cells. Transcriptome analysis showed that the impairment of intestinal function caused by OTA might be partly attributed to apoptosis, which is probably associated with downregulation of murine double minute 2 (MDM2) expression and upregulation of Noxa and caspase 3 (CASP3) expression. This study has highlighted the intestinal toxicity of OTA and provided a genome-wide view of biological responses, which provides a theoretical basis for enterotoxicity and should be useful in establishing a maximum residue limit for OTA.