Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons. To pump their substrates against steep concentration gradients, they utilize the energy stored in the form of electro-chemical gradients of ions, primarily sodium. Their mechanism involves transitions between extracellular- (outward-) and intracellular- (inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane. Our crystallographic studies on a bacterial homologue of glutamate transporters suggest that this process entails trans-membrane movements of three discrete transport domains within a trimeric scaffold. Using single-molecule fluorescence resonance energy transfer imaging, we have directly observed large-scale transport domain movements. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods. We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle. In addition, by means of X-ray crystallography, we have uncovered smaller local conformational transitions associated with substrate and ion binding in the outward- and inward-facing states of these transporters.