Glucose transporters, exemplified by the disease-related human proteins GLUT1-4, are essential for cellular uptake of glucose. GLUT1 is responsible for constant uptake of glucose, maintained at approximately 5 mM concentration in blood, into erythrocytes through facilitative diffusion. Inactivating mutations of GLUT1, resulting in compromised transport activities for glucose, are associated with diseases as a result of lack of energy supply to the brain. We determined the crystal structure of human GLUT1 at 3.2 angstrom resolution in the inward-open conformation. The structure allows accurate mapping and potential mechanistic understanding of disease-associated mutations in GLUT1. We also determined three related crystal structures of XylE, an E. coli homologue of GLUT1-4, in complex with D-xylose, D-glucose, and 6-Bromo-6-deoxy-D-glucose, at resolutions of 2.8, 2.9, and 2.6 angstrom, respectively. XylE was captured in an outward-facing, partially occluded conformation. Most of the important amino acids responsible for recognition of D-xylose or D-glucose are invariant in GLUT1-4, suggesting functional and mechanistic conservation. Structural comparison of GLUT1 and XylE provides the structural basis underlying alternating access for MFS proteins and allows examination of transport mechanisms by passive facilitators versus active transporters.