Glycine transporter GlyT1 is the main regulator of neuronal excitation and inhibition mediated by neurotransmitter glycine in the brain. Prolonging glycinergic signalling through selective inhibition of GlyT1 has been pursued extensively over the past two decades as a key strategy for the treatment of a broad range of neurological/psychiatric disorders including schizophrenia. GlyT1 inhibitors achieve antipsychotic and pro-cognitive effects against many symptoms of schizophrenia, however a successful drug candidate has to come. To elucidate structure-based mechanisms for inhibition and transport in GlyT1, we have investigated its complexes with a benzoylpiperazine chemotype inhibitor and substrate glycine. Using an inhibition state-specific sybody and a serial synchrotron crystallography (SSX) approach, we have determined the structure of GlyT1 at 3.4 Å resolution to reveal the selective inhibitor-bound state, adopting an inward-open conformation. More recently, we have determined the cryo-electron microscopy (cryo-EM) structure of GlyT1 at 3.3 Å resolution showing the glycine-bound inward-facing occluded conformation. The data unveil a dual nature of non-competitive inhibitors of functional transport exhibiting also competitive binding to the substrate binding site of glycine. The results provide detailed insight into the mechanism of glycine transport and reuptake inhibition and help re-evaluate efforts for the development of efficacious GlyT1 inhibitors.