ATP-binding cassette (ABC) transporters couple ATP hydrolysis to the uptake and efflux of solutes across the cell membrane in bacteria and eukaryotic cells. In bacteria, these transporters are important virulence factors because they play roles in nutrient uptake and in secretion of toxins and antimicrobial agents. In humans, many diseases, such as cystic fibrosis, hyperinsulinemia, and macular dystrophy, are traced to defects in ABC transporters. Recent advances in structural determination and functional analysis of bacterial ABC transporters, reviewed herein, have greatly increased our understanding of the molecular mechanism of transport in this transport superfamily.