Abstract GABA synthesis in astrocytes mediates tonic inhibition to regulate patho-physiological processes in various brain regions. Monoamine oxidase B (MAO-B) has been known to be the most important metabolic enzyme for synthesizing GABA from the putrescine degradation pathway. MAO-B converts N 1 -acetylputrescine to N 1 -acetyl-γ-aminobutyraldehyde and hydrogen peroxide (H 2 O 2 ). Putrescine acetyltransferase (PAT), also known as spermidine and spermine N 1 -acetyltransferase 1 (SAT1), has been thought to be a feasible candidate enzyme for converting putrescine to N 1 -acetylputrescine. However, it has not been rigorously investigated or determined whether PAT/SAT1 contributes to GABA synthesis in astrocytes. To investigate the contribution of PAT/SAT1 to GABA synthesis in astrocytes, we conducted sniffer patch and whole-cell patch experiments with gene silencing of PAT/SAT1 by Sat1 shRNA expression. Our results showed that the gene silencing of PAT/SAT1 significantly decreased the MAO-B-dependent GABA synthesis, which was induced by putrescine incubation, leading to decreased Ca 2+ -dependent release of GABA in vitro . Additionally, we found that, from the brain slice ex vivo , putrescine incubation induces tonic GABA inhibition in dentate gyrus granule cells, which can be inhibited by MAO-B inhibitor, selegiline. Consistent with our in vitro results, astrocytic gene silencing of PAT/SAT1 significantly reduced putrescine incubation-induced tonic GABA current, possibly by converting putrescine to N 1 -acetylputrescine, a substrate of MAO-B. Our findings emphasize a crucial role of PAT/SAT1 in MAO-B-dependent GABA synthesis in astrocytes.