Bacterial monoamine oxidases (MAOs) are FAD-dependent proteins catalyzing a relevant reaction for manyindustrial biocatalytic applications, ranging from production of enantiomerically pure building blocks forpharmaceutical synthesis to biosensors for monitoring food and beverage quality. The thermostable MAOenzyme from Thermoanaerobacterales bacterium (MAOTb) is about 36 % identical to both putrescine oxidase andhuman MAOs and can be efficiently produced in Escherichia coli. MAOTb preferentially acts on n-alkyl mono-amines but shows detectable activity also on polyamines and aromatic monoamines. The crystal structures ofMAOTb in complex with putrescine, benzylamine, spermidine and n-heptylamine at resolution ranging from 1.6to 2.3 Å resolution revealed the binding mode of substrates to the enzyme. The MAOTb active site is highlyconserved in the inner part of the cavity in front of the flavin ring (re face), where the presence of two tyrosineresidues creates the substrate amine binding site that is found also in human MAOs. Instead, more distantly fromthe flavin, the entrance of the catalytic site is much more open in MAOTb and features a different arrangement ofamino acids. Site-directed mutagenesis targeting residues Ala168, Thr199 and Val324 allowed the identificationof key residues in ligand binding to alter substrate specificity. The A168D variant showed a higher activity onputrescine than wild-type, whereas by replacing either Thr199 or Val324 to Trp a marked enhancement in kcat/KM values was found on n-alkyl-monoamines and on aromatic amines.