The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na+/H+ antiporters and an Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). The exact contribution of the various Na+/H+ antiporters to maintain Na+ and H+ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na+/H+ antiporter NhaA and the Na+-NQR are required by the bacteria for Na+ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na+ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na+/H+ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the PnhaB and PpepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na+ homeostasis in V. cholerae.