Natural food contaminants such as mycotoxins are an important problem for human health. Deoxynivalenol (DON) is one of the most common mycotoxin detected in cereals and grains. Its toxicological effects mainly concern the immune system and the gastrointestinal tract. This toxin is a potent ribotoxic stressor leading to MAPKinase activation and inflammatory response. DON frequently co-occurs with its glycosylated form, the masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G). The toxicity of this later compound remains unknown in mammals. This study aims to assess the ability of D3G to elicit a ribotoxic stress and to induce intestinal toxicity. The toxicity of D3G and DON (0-10µM) was studied in vitro, on the human intestinal Caco-2 cell line, and ex vivo on porcine jejunal explants. First, an in silico analysis revealed that D3G, contrary to DON, was unable to bind to the A site of the ribosome peptidyl transferase center, the main targets for DON toxicity. Accordingly, D3G did not activate JNK and P38 MAPKs in treated Caco-2 cells and did not alter viability and barrier function on cells, as measured by the trans epithelial electrical resistance. Treatment of intestinal explants for 4 hours with 10µM DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines as measured by qPCR and pan-genomic microarray analysis. By contrast, expression profile of D3Gtreated explants was similar to that of controls and these explants didn't show histomorphology alteration. In conclusion, our data demonstrated that glycosylation of DON suppress its ability to bind to the ribosome and decreases its intestinal toxicity.