The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2”)-Ia, which confers resistance to gentamicin, tobramycin and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2’’)-Ia enzyme. ANT(2”)-Ia confers resistance by magnesium-dependent transfer of nucleoside monophosphate (AMP) to the 2”-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg2+ ions, positioning of the modifiable substrate ring and presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2’’)-Ia has a two-domain structure with an N-terminal active site architecture that is conserved amongst other antibiotic nucleotidyltransferases including Lnu(A), LinB, ANT(4’)-Ia, ANT(4”)-Ib and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2”)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically-distinct molecules.