Deoxynivalenol-3-β-D-glucoside (D3G), a plant metabolite of the Fusarium mycotoxin deoxynivalenol (DON), might be hydrolyzed in the digestive tract of mammals, thus contributing to the total dietary DON exposure of individuals. Yet, D3G has not been considered in regulatory limits set for DON for foodstuffs due to the lack of in vivo data. The aim of our study was to evaluate whether D3G is reactivated in vivo by investigation of its metabolism in rats. Six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight (b.w.)) and the equimolar amount of D3G (3.1 mg/kg b.w.) by gavage on day 1, 8 and 15, respectively. Urine and feces were collected for 48 h and analyzed for D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) by a validated LC-tandem mass spectrometry (MS/MS) based biomarker method. After administration of D3G, only 3.7±0.7% of the given dose were found in urine in the form of analyzed analytes, compared to 14.9±5.0% after administration of DON, and only 0.3±0.1% were detected in the form of urinary D3G. The majority of administered D3G was recovered as DON and DOM-1 in feces. These results suggest that D3G is little bioavailable, hydrolyzed to DON during digestion, and partially converted to DOM-1 and DON-GlcA prior to excretion. Our data indicate that D3G is of considerably lower toxicological relevance than DON, at least in rats.