The mycotoxin deoxynivalenol (DON) is a chronic problem in cereals in temperate areas worldwide. Above regulatory levels, DON contamination can result in significant economic loss both to the primary producer and the feed industry in terms of increased costs. Here we report the enzymatic biotransformation of DON to a novel stable metabolite by a soil-borne strain of Bacillus subtilis. Proteomic analysis of activity-enriched protein fractions from this B. subtilis strain identified the glycosyltransferase YjiC as the enzyme responsible for the observed DON biotransformation. Liquid chromatography high-resolution tandem mass spectrometry and NMR spectroscopic analysis demonstrated that YjiC glycosylates DON at the 7-hydroxyl position, producing the novel metabolite DON-8,15-hemiketal-7-glucoside (HKDON7G). In toxicity experiments, duckweed exposed to 20 μM HKDON7G showed no phytotoxicity when compared to DON. Stability testing of HKDON7G demonstrated that it is significantly more resistant to enzymatic and microbial hydrolysis compared to DON-3-glucoside. This study is the first to report a chemical modification to the 7-hydroxyl position of DON and presents a novel mechanism for the detoxification of DON-contaminated food and feed.