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A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.