The glycine transporter GlyT2 regulates synaptic glycine concentrations to control inhibitory neurotransmission. Glycine transport inhibitors elevate synaptic glycine concentrations and provide analgesia in animal models of pain¹. We have sought to understand how glycine and GlyT2 selective inhibitors interact with the transporter. A crystal structure of a bacterial leucine transporter, LeuT, has been determined and we have used this model to better understand the structural basis for GlyT2 function. Mutations of residues in GlyT2 that correspond to the leucine binding site of LeuT change substrate selectivity of the transporter. For example, W482F changes GlyT2 from being highly selective for glycine to a promiscuous transporter that allows transport of Alanine, Valine, Leucine, Cysteine, Serine, Tyrosine and Phenylalanine. ALX1393 is a GlyT2 inhibitor that shows 20-fold selectivity over the closely related glycine transporter GlyT1. Inhibition of GlyT2 by ALX1393 causes a reduction in maximal rate of glycine transport and also increases the K_0.5 for glycine demonstrating that it has characteristics of both a competitive and non-competitive inhibitor. Thus, the binding site for ALX1393 may overlap with the glycine binding site, but other regions may also contribute to the binding site. 7 mutations, that alter substrate selectivity of GlyT2, cause 4-20 fold reductions in potency of ALX1393 inhibition confirming the overlap in substrate and inhibitor binding sites. The outward facing conformation of LeuT contains an aqueous accessible vestibule, which has been proposed to form a low affinity binding site for non-competitive inhibitors. We introduced a variety of mutations in this vestibule that may have the capacity to interact with ligands, but none of the 10 mutations caused any change in the potency of ALX1393 inhibition. We conclude that ALX1393 binds to the substrate binding site of GlyT2 and that the external vestibule does not contribute to the inhibitor binding site.