Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug-resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N-acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high-resolution crystal structures of Mabs_Eis2 in its apo- and cofactor-bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high-resolution crystal structures presented here may pave the way to the design of Eis2-specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. DATABASE: Structural data are available in the PDB database under the accession numbers: 6RFY, 6RFX and 6RFT.,© 2019 Federation of European Biochemical Societies.