AbstractSecondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters’ energy landscapes might be a widespread route of their functional diversification.