Deoxynivalenol (DON), a mycotoxin prevalent in grains and feeds, poses a serious threat to humans and animal health. The intestine is the primary target organ of DON toxicity. However, the molecular mechanisms linking DON exposure to intestinal injury remain poorly understood. Here, we identify DEK, a multifunctional nuclear protein, is a critical target protein of DON-induced intestinal damage through endoplasmic reticulum (ER) stress activation. We demonstrate that DON directly binds to DEK via key residues (Phe56, Lys68, and Leu106) and disrupts its interaction with IRE1α, a central regulator of the ER stress. Structural analyses revealed that Lys68 of DEK is essential for its binding to IRE1α. DON interferes with the interaction between DEK and IRE1α by binding to DEK and reducing its expression. DEK overexpression attenuated ER stress and inflammatory cytokine production (, , ) in intestinal cells. Conversely, DEK knockdown exacerbates DON-triggered ER stress by activating the IRE1α/PERK/eIF2α pathways, thereby amplifying intestinal inflammation via MAPK signaling. In vivo, DON-treated mice and piglets exhibited intestinal villus atrophy, crypt hyperplasia, and DEK-dependent ER stress activation. Collectively, our findings reveal a novel DEK-IRE1α axis in DON toxicity, underscoring that DEK may serve as a potential therapeutic target for mitigating DON-induced intestinal injury.