The 18-kDa translocator protein (TSPO) is evolutionarily conserved from bacteria to humans. TSPO expression has been observed in essentially all mammalian tissues, with a preferential localization in the outer mitochondrial membrane. TSPO is involved in various physiological functions. The evidence suggests that TSPO may function in different protein complexes. In mammalian cells, the best-characterized activity of TSPO is the transport of cholesterol from the cytosol to the mitochondrial matrix, where cholesterol is converted to a precursor of steroids or bile salts. No atomic structure of TSPO is currently available. TSPO does not belong to any known membrane protein structural family. It has five transmembrane domains containing α-helices that are involved in the transport of cholesterol. Cytosolic loops are involved in ligand binding and the activation of transport. It has been suggested that TSPO could form homopolymers within heteropolymer complexes. Because of the low native abundance of TSPO, production of recombinant TSPO is a first step for any structural study. In this chapter, we present the current understanding of TSPO overexpression studies in bacteria, purification of functional TSPO, and different approaches to solve TSPO structure.