Secondary active membrane transporters use the energy of electrochemical ion gradients to concentrate their substrates. Transporters within the same family often evolve to use different ions depending on physiological needs or bioavailability. How such functional differences arise despite similar three-dimensional protein structures is mostly unknown. We used structural biology, phylogenetics, and ancestral sequence reconstructions to explore how ion coupling mechanisms expanded in human glutamate transporters to achieve extraordinary concentrative power and how prokaryotic homologs evolved to use either sodium or proton gradients. We found that the evolutionary switch from sodium to proton coupling occurred via an intermediate clade. The reconstructed intermediate ancestral transporter features intact sodium-binding sites but no longer requires ions to bind its substrate. Thus, the evolution from sodium- to proton-coupling might have proceeded through an ion-independent intermediate. Overall, we show that changes in ion coupling could be due to mutations in ion-binding sites or distant allosteric mutations altering the energy landscape of the transporter. We also show how phylogenetic analyses and ancestral sequence reconstructions can be used to guide protein mechanistic studies.