Ischemic stroke is the second leading cause of death worldwide. Compelling evidence demonstrates that following an ischemic event, neuronal death is triggered by uncontrolled glutamate release leading to overactivation of glutamate sensitive N-methyl-D-aspartate receptor (NMDAR). For gating, NMDARs require not only the binding of glutamate, but also the binding of glycine or a glycine-like compound as a co-agonist. Glycine fulfills several roles in biology including protein synthesis, inhibitory transmission via glycine receptor activation and excitatory transmission through NMDARs. Low glycine doses enhance NMDAR function while high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. The physiological relevance of GINI has been questioned given that the high-affinity glycine transporter type 1 (GlyT1), located on astrocytes and neurons, maintains synaptic glycine concentrations far below the level that would saturate the glycine binding site (GBS). Here, we report that following an ischemic event, in vivo, GINI also occurs and provides neuroprotection. Mice pre-treated with a GlyT1 antagonist (GlyT1-A), which increases glycine synaptic levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioural deficits following stroke induction by either photothrombosis or endothelin-1. Therefore, increasing glycine levels in the synaptic cleft will enhance GBS occupancy and will reach the set point to trigger GINI. However, we report that GINI is triggered during ischemic stroke, in vivo, only in the presence of GlyT1-As. Moreover, we show evidence that in ischemic conditions, GlyT1-As preserve the vasculature in the peri-infarct area. Therefore, the clinical efficacy of GlyT1-As should be tested for the treatment of ischemic stroke.