Aminoglycoside antibiotics are well-known inhibitors of bacterial protein synthesis, which act mainly by inhibiting ribosomal translocation and inducing miscoding errors. Arbekacin (ABK) is a semisynthetic aminoglycoside that was developed by adding a 3-amino-2-hydroxybutyric (AHB) moiety to the 2-deoxystreptamine (2-DOS) ring of dibekacin for counteracting the problem of enzyme-mediated resistance of aminoglycosides. Here, we have systematically compared the inhibition efficacy of ABK with other aminoglycosides by in vivo MIC determination and in vitro fast-kinetics based translocation and termination assays complemented with a high-resolution cryo-EM structure. ABK presents significantly lower MIC value compared to its parent antibiotics kanamycin and dibekacin. Consistent with that, ABK inhibits translocation with lower inhibition constant and reside on the ribosome for significantly longer time than the classical aminoglycosides. Our 3.1 Å resolution cryo-EM structure of ABK-bound ribosome containing mRNA and initiator-tRNA, shows interactions of the unique AHB moiety of ABK with rRNA nucleobases, which likely provide additional stabilization of ABK at the canonical aminoglycoside binding pocket and contribute to its prolonged dwelling time. Our structural and functional analyses provide molecular basis for higher potency of ABK in bacterial translation inhibition and opens the possibility of rational design of new antibiotics.