We sought to review the potential role of isepamicin against infections with contemporary Gram-negative bacteria. We searched PubMed and Scopus databases to identify relevant microbiological and clinical studies published between 2000 and 2010, and we retrieved 11 and three studies, respectively. A total of 4901 isolates were examined in the in vitro studies. Isepamicin had higher in vitro activity compared with amikacin in four studies, was as active as amikacin in six studies and in the remaining study both were inactive. Regarding specifically the studies that included multidrug-resistant bacteria, isepamicin appeared superior to amikacin in two studies, as active as amikacin in one study and both did not exhibit activity in one study. In the clinical studies, isepamicin was as active as amikacin for the treatment of 55 children with urinary tract infections. In conclusion, isepamicin might be active in vitro against Gram-negative bacteria with resistance to amikacin and other aminoglycosides.