Cellular homeostasis relies on solutes crossing cell membranes against their concentration gradients. Membrane transporters catalyze this energetically unfavorable movement using energy from the downhill movement of ions. Often, related transporters couple to either Na+ or H+ gradients; it can be difficult to glean the mechanistic reasons between such differences because the ability to use ions with such different physicochemical properties might require many changes in the transporter. Since evolutionary adaptations to environmental constraints such as salt concentrations and pH likely gave rise to the switch between sodium- and proton-motive forces, we hypothesized that phylogenetic reconstruction of ion-coupling may be a method to study such complex changes. We used phylogenetic analysis to reconstruct the evolution of prokaryotic glutamate transporters and deduce stepwise amino acid sequence changes resulting in the emergence of H+-coupled transporters from Na+-coupled ancestors. The analysis suggests that in addition to changing protonatable residues, introducing H+-dependence increases the hydrophobicity of previously polar regions proximal to the substrate and Na+ binding sites. To contextualize these changes, we used ancestral sequence reconstruction (ASR) to infer transporter sequences before and after the emergence of H+-coupling. We expressed and purified the inferred ancestral transporters, enabling their biochemical characterization and high-resolution cryo-EM structures. Surprisingly, the ancestral transporter before the emergence of H+-coupling shows Na+-independent substrate binding even though its Na+-binding pockets are structurally near-identical to Na+-coupled homologs. The emergence of H+-coupling preserves the protein architecture while subtly restructuring these critical regions. Our results suggest that changes in local structure and complex global allosteric properties underlie the diversification of the ion-motive forces used within the glutamate transporter family. The approach we developed might be broadly applicable to dissecting complex allosteric properties of membrane proteins.