In this study we provide the first biochemical characterization of a bacterial pyranose 2-oxidase (AsP2Ox) from Arthrobacter siccitolerans. The enzyme catalyzes the oxidation of several aldopyranoses at the C-2 position, coupling it to the reduction of dioxygen to hydrogen peroxide. Pyranose 2-oxidases belong to the glucose-methanol-choline oxidoreductase family. A structural model based on the known X-ray structure of P2Ox from Phanerochaete chrysosporium supports that AsP2Ox shares structural features with well-characterized fungal P2Oxs. The gene coding for AsP2Ox was cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme is a 64-kDa monomer containing a non-covalently bound flavin adenine dinucleotide (FAD) cofactor, distinct features as compared with fungal counterparts that are ∼ 270kDa homotetramers with covalent-linked FAD. AsP2Ox exhibits a redox potential of -50mV, an optimum temperature of 37C and an optimum pH at 6.5. AsP2Ox oxidizes d-glucose at the highest efficiency, using additionally d-galactose, d-xylose, l-arabinose and d-ribose as electron donors, coupling their oxidation to the reduction of both dioxygen and 1,4-benzoquinone. AsP2Ox shows a relatively low thermal stability with a melting temperature (T m ) of 43C and a half-life (t 1/2 ) at 40C of 25min. This work expands the repertoire of bacterial oxidoreductases with importance in biotechnological and diagnostic applications.