Methylglyoxal (MG) is a neurotoxic α,β- dicarbonyl derived from glycolysis. MG is detoxified by the ubiquitous, cytosolic glyoxalase (Glo) system. In the present work the temporal and spatial changes of Glo1 after acute cerebral lesions and the effects of its inhibitor ethyl pyruvate (EP) on astrocytic scar formation have been analyzed. Western blot analyses of excitotoxically lesioned organotypic hippocampal slice cultures (OHSC) revealed a temporal redistribution of Glo1 monomer and dimer, favoring a Glo1 dimer increase. Additionally, a translocation of Glo1 to the neuronal cell membrane and enhanced Glo1 immunoreactivity after excitotoxic insult were observed. Immunohistochemistry subsequent to permanent middle cerebral artery occlusion (pMCAO) showed a predominantly neuronal Glo1 immunoreactivity that was no longer visible after 60 days. Astrocytes displayed the highest Glo1 immunoreactivity 60 days after injury. Glo1 immunoreactivity was neither changed at the investigated time points nor after LPS or EP stimulation in BV2 and HT22 cells. EP decelerated astrocytic scar formation in the scratch wound assay.