Deoxynivalenol (DON) is one of the most common mycotoxins in cereals and their by-products. Its adverse effects on animal and human health have been extensively studied in the intestine, but little attention has been paid to another target organ for mycotoxins, the liver that is potentially exposed after intestinal absorption and enterohepatic circulation. To assess DON's toxicity in an ex vivo model structurally and physiologically closer to the whole liver, we developed a pig precision-cut liver slices (PCLS) model. PCLS contain all cell types and maintain intercellular and cell-matrix interactions, among other architectural features of the liver. The human HepG2 cell line was used for comparison. We observed that after a short exposure, DON reduced the cell viability of HepG2 cells and induced the expression of genes involved in apoptosis, inflammation and oxidative stress. When PCLS were exposed to DON, damage to the tissues was observed, with no changes in markers of liver function or injury. Exposure to the toxin also triggered liver inflammation and apoptosis, effects already observed in pigs fed DON-contaminated diets. Overall, these data demonstrate that DON had toxic effects on a liver cell line and on whole liver tissue, consistent with the effect observed during in vivo exposure. They also indicate that pig PCLS is a relevant and sensitive model to investigate the liver toxicity of food contaminants.