The unique pharmacokinetic and pharmacodynamic activities of glycopeptide antibiotics are conferred by tailoring steps occurring on the aglycone. Here, we report that protein Dbv29, involved in the biosynthesis of A40926, is a novel flavin mononucleotide-dependent primary alcohol glycopeptide hexose oxidase that carries out a four-electron oxidation reaction. Dbv29 catalyzes the last step in a multistep sequence to complete N -acyl aminoglucuronic acid substituent biosynthesis for a potent drug lead. The characterized enzyme may provide a new way to enhance the efficacy of currently used glycopeptide drugs. This detailed function-mechanism analysis of the enzyme increases our knowledge of this new class of enzyme.