The neuronal glycine transporter 2 (GlyT2) belongs to the large SLC6 family of Na+/Cl--dependent neurotransmitter transporters. At its extreme C-terminus, GlyT2 carries a type III PDZ domain binding motif (PDZ-ligand motif), which interacts with the PDZ domain protein syntenin-1. Here, we investigated the physiological role of the GlyT2 PDZ-ligand motif by a loss-of-function approach. Inactivation of the PDZ-ligand motif did not impair the localization, glycosylation and transport function of recombinant GlyT2 expressed in HEK293T cells. However, in transfected hippocampal neurons, the synaptic localization of GlyT2 was significantly reduced upon PDZ-ligand motif inactivation. Co-localization of GlyT2 with marker proteins of excitatory and inhibitory synapses was decreased by down to 50% upon PDZ-ligand motif deletion as compared to the wild-type protein. These data indicate that the C-terminal PDZ-ligand motif of GlyT2 plays an important role in transporter trafficking to and/or stabilization at synaptic sites.