Since the discovery of M. tuberculosis, the nutritional requirements for the growth of mycobacteria have been extensively studied, while the essential nutrients for M. tuberculosis inside the human host remain unclear. The UgpABCE transporter of M. tuberculosis is one of ﬁve putative permeases for carbohydrate uptake, and is genetically predicted to be a sn-glycerol 3-phosphate (sn-G3P) importer. UgpB is a periplasmic binding protein, which has been reported to be a promising vaccine candidate against TB. The crystal structure of M. tuberculosis UgpB (MtbUgpB) in 1.5Å resolution was determined. The overall structure of MtbUgpB is similar to its Escherichia coli homolog, but differences exist in the substrate-binding pocket. The most striking difference is the substitution of Trp169 of E. coli by Leu205 of M. tuberculosis. Among the binding candidates of UgpB, MtbUgpB showed a preference for glycerophosphocholine (GPC), but had no detectable binding affinity for sn-G3P, G2P and maltose. Mutation of Leu205 abolishes GPC binding, suggesting that Leu205Ala is a determinant for GPC binding, yet mutation of Leu205Trp, which resembles the binding pocket of E. coli UgpB, showed no detectable binding activity for G3P. As formation of a complex between the protein and the substrate is a prerequisite for ABC importers to transport nutrients across membranes, our result suggests that GPC, instead of the predicted sn-G3P, is the ligand that M. tuberculosis UgpB accepts for transport into the cytosol, and provides the main carbon and phosphate sources for growth and survival of M. tuberculosis inside the human host.