AbstractCarbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase fromHerbinix hemicellulosilytica,HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domainHhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperatureTmof 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan.HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain ofHhXyn5A did not demonstrate detectable activity.HhXyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homologCtXyn5A,HhXyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling ofHhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5concluded that the active site ofHhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities betweenHhXyn5A andCtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.