Copper amine oxidases are important for the metabolism of a range of biogenic amines. Here, we focus on substrate specificity in the E. coli copper amine oxidase (ECAO) and specifically the role of Tyr 381. This residue, and its equivalent, in other copper amine oxidases has been referred to as a "gating" residue able to move position depending upon the presence or absence of amine substrate. The position of this residue suggests a role in substrate selectivity. We have compared the properties of two variant forms of ECAO, Y381F and Y381A, with wild-type enzyme by steady-state kinetics of oxidation of a number of amine substrates, modes of inhibitor interactions and X-ray structure determination. Y381F displays a similar catalytic efficiency to wild type against the preferred substrate β-phenylethylamine. In both cases oxidation of the alternative aromatic amine substrate benzylamine is relatively poor, although Y381F represents an efficient benzylamine oxidase. By contrast, Y381A performed poorly against both aromatic substrates predominantly due to an increased K (M) which we propose is due to the lack of an aromatic residue to orient substrate towards the TPQ and active site base. These results are supported by different behaviour of Y381A to inhibition with 2-hydrazinopyridine. We also report on methylamine turnover by the three enzymes. We propose that Y381, together with another residue Y387, may be considered of critical importance for the substrate selectivity of ECAO, through stacking or hydrophobic interactions with substrate.