Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells.