Overexpression, purification, and crystallization of eukaryotic membrane proteins represent a major challenge for structural biology. In recent years, we have solved the crystal structures of the human glucose transporters GLUT1 in the inward-open conformation at 3.17 Å resolution and GLUT3 in the outward-open and occluded conformations at 2.4 and 1.5 Å resolutions, respectively. Structural elucidation of these transporters in three distinct functional states reveal the molecular basis for the alternating access transport cycle of this prototypal solute carrier family. It established the molecular foundation for future dynamic and kinetic investigations of these GLUTs, and will likely facilitate structure-based ligand development. In this chapter, we present the detailed protocols of recombinant protein expression, purification, and crystallization of GLUT1 and GLUT3, which may help the pursuit of structural elucidation of other eukaryotic membrane proteins.