The cation/proton antiporter Vc-NhaP2 from the important human pathogen Vibrio cholerae acts as a K+/H+ exchanger in vivo, contributing to the survival of V. cholerae at low pH and possibly enhancing the chances of the ingested V. cholerae cells to pass the acidic gastric barrier. Vc-NhaP2 is also able to exchange Na+ but not Li+ ions for H+. We attempted to identify the amino acids responsible for cation specificity of Vc-NhaP2 by limited alanine-scanning mutagenesis. Here we report a novel mutation, Gly159Ala, which enables Vc-NhaP2 to exchange Li+ for H+. Substitutions of Gly159 with Leu, Asp, or Lys, but not Ser had the same effect. Alanine substitution of the highly conserved Asn161 or Asp162 residues located nearby resulted in total inactivation of the antiporter. In the course of alanine-scanning mutagenesis, two distant residues, Asp273 and Leu289 that control the K+ /Na+ selectivity of Vc-NhaP2 were also identified. These findings support the idea of “ligand shading” in the active site of Vc- NhaP2, where different alkali cations are coordinated by overlapping but not identical sets of ligands, thus differently affecting the probability of protonation of the antiporter during the catalytic cycle. In a phylogenetic context, our results demonstrate one of the mechanisms underlying rapid divergent evolution of paralogous membrane transporters through the accumulation of seemingly insignificant single-point mutations (such as Gly-to-Ala) that might, nevertheless, have an immediate adaptive value.