The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis. Eis from M. tuberculosis (Eis-Mtb) and M. smegmatis (Eis-Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis-Mtb is divided into two narrow channels, whereas Eis-Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis-Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis-Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis-Mtb and for the functional differences from Eis-Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis-Mtb by mutating them into their Eis-Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis-Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis-Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis-Mtb and Eis-Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis-Mtb into Ala plays a role in APR acetylation.