Deoxynivalenol (DON) is a potent mycotoxin produced by Fusarium molds and affects intestinal nutrient absorption and barrier function in experimental and farm animals. Free DON and the plant metabolite DON-3-β-d-glucoside (D3G) are frequently found in wheat and maize. D3G is stable in the upper human gut, but some human intestinal bacteria release DON from D3G in vitro. Furthermore, some bacteria derived from animal digestive systems degrade DON to a less toxic metabolite, deepoxy-deoxynivalenol (DOM-1). The metabolism of D3G and DON by the human microbiota has not been fully assessed. We therefore conducted in vitro batch culture experiments assessing the activity of the human fecal microbiota to release DON from D3G. We also studied detoxification of DON to DOM-1 by the microbiota and its potential effect on urinary DON excretion in humans. Fecal slurry from five volunteers was spiked with DON or D3G and incubated anaerobically (from 1 h to 7 days), and mycotoxins were extracted into acetonitrile. Mycotoxins were detected in fecal extracts and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The fecal microbiota released DON from D3G very efficiently, with hydrolysis peaking after 4 to 6 h. The fecal microbiota from one volunteer transformed DON to DOM-1. Urine from the same volunteer also contained DOM-1 (4.7% of DON), whereas DOM-1 was not detectable in urine from other volunteers. Our results confirm that the fecal microbiota releases DON from its glycosylated form, hence increasing the toxic burden in exposed individuals. Furthermore, this is first evidence that the human fecal microbiota of one volunteer detoxifies DON, resulting in the appearance of DOM-1 in urine.