It is known that brain activity is fueled by glucose, but the identity of the cell type that metabolises the sugar remains elusive. In order to address the issue, we have developed transport assays that allow single-cell real-time measurement of glucose transport in mixed rat hippocampal cultures using confocal microscopy. Our data shows that the neurotransmitter glutamate triggers opposite responses in astrocytes and neurons. Astrocytes, which only express GLUT1, responded with a two to three-fold stimulation of the uptake rate of the hexoses 6-NBDG, 2-NBDG and galactose. The effect was due an increase in the Vmax for uptake. The rate of efflux was unaffected, which using the ″simple carrier″ model can be accounted economically by a decrease in the asymmetry of the transporter. Strikingly, neurons responded with a > 80% inhibition of the hexose uptake rate. The IC50 was 5 μM, which is well in the range estimated for glutamate in the synaptic cleft. The phenomenon in neurons was mimicked by AMPA and was abolished pharmacologically by CNQX and functionally by removal of extracellular sodium, so it is seems to be mediated by AMPA receptors. Both astrocytic stimulation and neuronal inhibition of hexose uptake rates by glutamate were detected in the range of seconds and reverted upon glutamate removal We conclude that glutamate redistributes glucose towards astrocytes and away from neurons. These phenomena provide novel regulatory nodes for brain energy metabolism and support a model in which blood-borne glucose is first metabolised by astrocytes and then transferred to active neurons as a more oxidized form, perhaps lactate. Were these mechanisms to be functional in the brain, they would mean that functional imaging by FDG-PET scanning reports an astrocytic phenomenon, although directly linked to neuronal activity by means of glutamate release.