The ABC transporter MsbA is an integral membrane protein involved in the transport of lipid A and lipopolysaccharides to the outer leaflet of the inner membrane in bacteria. Here, the critical role of the natural substrate lipopolysaccharide in the crystallization and diffraction quality of MsbA crystals is reported. Initial crystals grown in complex with ATP-vanadate alone diffracted to approximately 9 A. Screening of the natural substrate lipopolysaccharides led to the crystallization of MsbA in complex with ADP-vanadate and Ra lipopolysaccharide. The increased order within the crystal lattice allowed structure determination to 4.2 A.