Glucose is the primary fuel to life on earth. Cellular uptake of glucose is a fundamental process for metabolism, growth, and homeostasis. The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, are prototypes in the study of solute transport. Debilitating mutations and mis-regulation of GLUTs are associated with a broad spectrum of human disease. We were able to determine the atomic structures of human GLUTs in multiple conformations during a transport cycle, which reveal the molecular basis for ligand recognition and transport. The crystal structures allow accurate mapping and potential mechanistic understanding of disease-associated mutations in GLUT1 and lay out a foundation for rational ligand design.