Alzheimer's disease (AD) leads to cerebral accumulation of insoluble amyloid-β plaques causing synaptic dysfunction and neuronal death. Neurons rely on astrocyte-derived glutamine for replenishment of the amino acid neurotransmitter pools. Perturbations of astrocyte glutamine synthesis have been described in AD, but whether this functionally affects neuronal neurotransmitter synthesis is not known. Since the synthesis and recycling of neurotransmitter glutamate and GABA are intimately coupled to cellular metabolism, the aim of this study was to provide a functional investigation of neuronal and astrocytic energy and neurotransmitter metabolism in AD. To achieve this, we incubated acutely isolated cerebral cortical and hippocampal slices from 8-month-old female 5xFAD mice, in the presence of C isotopically enriched substrates, with subsequent gas chromatography-mass spectrometry (GC-MS) analysis. A prominent neuronal hypometabolism of [U-C]glucose was observed in the hippocampal slices of the 5xFAD mice. Investigating astrocyte metabolism, using [1,2-C]acetate, revealed a marked reduction in glutamine synthesis, which directly hampered neuronal synthesis of GABA. This was supported by an increased metabolism of exogenously supplied [U-C]glutamine, suggesting a neuronal metabolic compensation of the reduced astrocytic glutamine supply. In contrast, astrocytic metabolism of [U-C]GABA was reduced, whereas [U-C]glutamate metabolism was unaffected. Finally, astrocyte de novo synthesis of glutamate and glutamine was hampered, whereas the enzymatic capacity of glutamine synthetase for ammonia fixation was maintained. Collectively, we demonstrate that deficient astrocyte metabolism leads to reduced glutamine synthesis, directly impairing neuronal GABA synthesis in the 5xFAD brain. These findings suggest that astrocyte metabolic dysfunction may be fundamental for the imbalances of synaptic excitation and inhibition in the AD brain.