The energy-coupling factor (ECF) transporters, responsible for vitamin uptake in prokaryotes, are a unique family of membrane transporters. Each ECF transporter contains a membrane-embedded, substrate-binding protein (known as the S component), an energy-coupling module that comprises two ATP-binding proteins (known as the A and A'2 components) and a transmembrane protein (known as the T component). The structure and transport mechanism of the ECF family remain unknown. Here we report the crystal structure of RibU, the S component of the ECF-type riboflavin transporter from Staphylococcus aureus at 3.6-Å resolution. RibU contains six transmembrane segments, adopts a previously unreported transporter fold and contains a riboflavin molecule bound to the L1 loop and the periplasmic portion of transmembrane segments 4-6. Structural analysis reveals the essential ligand-binding residues, identifies the putative transport path and, with sequence alignment, uncovers conserved structural features and suggests potential mechanisms of action among the ECF transporters.