Deoxynivalenol (DON) is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans. Phenethyl isothiocyanate (PEITC) in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal. In the current study, we aimed to explore the effects of PEITC on DON-induced apoptosis, intestinal tight junction disorder, and its potential molecular mechanism in the porcine jejunum epithelial cell line (IPEC-J2). Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity, decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels, and increasing zonula occludens-1 (ZO-1), occludin and claudin-1 mRNA/protein expression. Meanwhile, PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) mRNA levels and intracellular reactive oxygen species (ROS) level, and a decrease of glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), catalase (CAT) and heme oxygenase 1 (HO-1) mRNA levels. Additionally, PEITC treatment significantly down-regulated autophagy-related protein 5 (ATG5), beclin-1 and microtubule-associated protein 1 light chain 3B (LC3-Ⅱ) mRNA/protein levels, decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3 (GFP-LC3) puncta and phosphatidylinositol 3 kinase (PI3K) protein expression, and up-regulated phospho-protein kinase B (p-Akt) and phospho-mammalian target of rapamycin (p-mTOR) protein expression against DON. However, the activation of autophagy by rapamycin, an autophagy agonist, abolished the protective effects of PEITC against DON-induced cytotoxicity, apoptosis and intestinal tight junction disorder. Collectively, PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROS-mediated autophagy.