The bacterial melibiose permease (MelB) belongs to the glycoside- pentoside- hexuronide:cation symporter family (GPH), a part of the major facilitator superfamily (MFS). Structural information regarding GPH transporters and other Na +-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the 3D crystal structures of Salmonella typhimurium MelB St in two conformations, representing an outward partially occluded and an outward inactive state of MelB St. MelB adopts a typical MFS fold, and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na +, Li +, or H +, which is in close proximity to the sugar-binding site. Both co-substrate-binding sites are mainly contributed by the residues from the N-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na +/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalyzed by MFS permeases in general.