An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s⁻¹ x 10⁻¹, Km 1.4 mM and Vmax 95 Umg⁻¹ protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzyme's active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.