Resistance to aminoglycoside antibiotics is mediated by chemical modifications: O-phosphorylation (APH enzymes), N-acetylation (AAC enzymes) and N-adenylation (ANT enzymes). In this entry, we present the structure of ANT(4')-IIb, an enzyme which adenylates tobramycin and amikacin antibiotics. The structure is dimeric and contains a nucleotidyltransferase domain. The antibiotic is bound in an electronegative pocket. This is only the second ANT enzyme structure and will be useful to understand the substrate discrimination mechanism and for identification of small molecule inhibitors.