Staphylococcus aureus confers life-threatening and difficult to treat infections worldwide and antimicrobial resistance is an increasing concern. While acquired resistance has been widely studied, little is known of the contributions from chromosomal determinants that upon inactivation may reduce the susceptibility towards antibiotics. The aim of this study was to identify genetic determinants that upon inactivation reduce aminoglycoside susceptibility in Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for reduced susceptibility to gentamicin. 9 mutants were confirmed by E-test to display between 2 and 16-fold reduced susceptibility to this antibiotic. All of the identified genes were associated with the electron transport chain and energy metabolism. Four mutant strains (menD, hemB, aroC and SAUSA300_0355) conferred the largest increase in gentamicin susceptibility and exhibited a small colony variant phenotype, whereas the remaining mutants (qoxA, qoxB, qoxC, ndh and hemX) displayed colony morphology similar to the wild type. All of the mutants, except hemX, displayed reduced membrane potential suggesting that reduced uptake of gentamicin is the predominant mechanism leading to reduced susceptibility. The results of this study demonstrate that Staphylococcus aureus possess multiple genes, which upon inactivation by mutagenesis reduce the membrane potential and thereby reduce the lethal activity of gentamicin.