The excitatory amino acid transporters (EAATs) are membrane transport proteins that clear neurotransmitter glutamate from the synaptic cleft and terminate neurotransmission. The clearance of glutamate is critical for brain functions because accumulated glutamate in the synaptic cleft is toxic for neuronal cells, and ultimately induces brain damage. Consistently, malfunction of EAATs is associated with brain disease such as epilepsy, ataxia and neurodegeneration. To transport glutamate into cytoplasm, EAATs harness free energy stored in the form of ionic electrochemical gradients across the membrane. They couple transport of one glutamate to symport of three sodium ions and a proton, and to antiport of a potassium ion. Many elucidated aspects of the mechanism of ion-coupled glutamate transport are based on studies of its archaeal homologue, GltPh, a sodium-coupled aspartate transporter from Pyrococcus horikoshii. GltPh is a great structural and functional framework to understand sodium-substrate coupling in the family of glutamate transporters. First, like EAATs, GltPh couples transport of one aspartate to symport of three sodium ions. Second, GltPh has sequence identity with EAATs of ca 37% overall, and higher homology in sodium and substrate binding sites. Third, crystal structures of GltPh in key functional states have been determined to high resolutions. It has been shown that sodium and aspartate binding to GltPh are tightly coupled. In the absence of sodium, aspartate shows no appreciable affinity to GltPh, but in the presence of millimolar sodium concentrations, aspartate binds with nanomolar affinity. However, the molecular mechanism of the tight sodium-aspartate coupling remains unclear. To investigate the sodium- coupled mechanism, we crystallized GltPh in apo, sodium-bound and sodium/aspartate-bound states. Comparison of the structures shows that sodium binding induces exposure of substrate binding site to the solvent upon opening of the extracellular gate and a conformational change of a highly conserved motif in substrate binding site, which is important in both aspartate and sodium binding. In addition, we found a new cation binding site, which may serve as a potassium binding site in EAATs. We further investigated how sodium ion binding affects ligand release and binding to GltPh in a cytoplasmic-facing conformation. The kinetics of inhibitor binding, which mimics the earlier events of substrate binding, suggests that binding follows a sodium-dependent ‘conformational selection’ mechanism, whereby rate-limiting binding of the first sodium ion stabilizes conformation of the transporter competent to bind substrate. The kinetics of ligand dissociation suggests that dissociation of one or two sodium ions precedes and determines the rate of ligand dissociation. Overall, aspartate binding kinetics suggest that sodium ion bindings before and after controls the rate of aspartate binding and dissociation.