Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and Gram-negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Antibiotic resistance is a growing problem for all classes of anti-infective agents. One of the first groups of antibiotics to encounter the challenge of resistance was the aminoglycoside -aminocyclitol family. Initially, the resistance that emerged in organisms such as Mycobacterium tuberculosis was restricted to modification of the antibiotic targets, which we now know to be the bacterial ribosomal rRNA and proteins. As new aminoglycosides came to the clinic, however, the prevalence of chemical modification mechanisms of resistance became dominant. Enzymatic modification of aminoglycosides through kinases (O-phosphotransferases, APHs), Oadenyltransferases (ANTs) and N-acetyltransferases (AACs) has emerged in virtually all clinically relevant bacteria of both Gram-positive and Gram-negative origin. Although their clinical use has been extensive, their toxicity and the prevalence of resistance in clinical strains have prompted the pharmaceutical industry to look for alternatives. Whereas the search for novel targets for antibiotics from the genomic information is ongoing, no antibacterial agent based on these efforts has so far entered clinical trials. Meanwhile, structural knowledge of the ribosome, the target for aminoglycosides, has invigorated the field of antibiotic development. It is expected that knowledge of the binding interactions of aminoglycosides and the ribosome would lead to concepts in drug design that would take us away from the parental structures of aminoglycosides in the direction of different structural classes that bind to the same ribosomal target sites as aminoglycosides. The challenge to ensure the continued use of these highly potent antibacterial agents will require the effective management of resistance at several levels. One potential mechanism of circumventing resistance is the development of inhibitors of modification enzymes, a methodology that is now well established in the β-lactam field. This approach requires knowledge of resistance at the molecular and atomic levels for the rational design of inhibitory molecules. The understanding of the molecular basis for aminoglycoside resistance modification has been greatly enhanced by the recent availability of representative 3D-structures from the three classes of modifying enzymes: kinases, acetyltransferases and adenyltransferases. The challenge is now to firmly establish the mechanisms of enzyme action and to use this information to prepare effective and potent inhibitors that will reverse antibiotic resistance. In this review, we discuss the molecular mechanisms of resistance of aminoglycosides specifically on aminoglycoside-modifying enzymes and newly developed strategies to circumvent resistance including antisense technology, which is an example of new strategy to deal with antibiotic resistance.