Gluconeogenesis is a well-established metabolic process whereby glucose is generated from small carbon molecules in the liver and kidney to maintain blood glucose levels. Expression of gluconeogenic genes has been reported in other organs of mammals and insects, where their function is not yet known. In the fruit fly, one of the gluconeogenic genes, glucose-6-phosphatase (G6P) is exclusively expressed in the CNS. Using a fluorescence resonance energy transfer (FRET)-based glucose sensor, we show that a small subset of neurons in the fly brain is capable of carrying out gluconeogenesis. Moreover, we find that G6P mutant flies exhibit low whole-body glucose levels within 24 h of food deprivation. This phenotype can be mimicked by silencing G6P neurons and rescued by experimentally controlled activation in the absence of G6P. These results indicate that neural activity of G6P neurons, but not glucose production per se, is critical for glucose homeostasis. Lastly, we observe that neuronal gluconeogenesis promotes anterograde neuropeptide distribution from the soma to axon terminals, suggesting that the generation of glucose facilitates neuropeptide transport. Together, our analysis reveals a novel role for gluconeogenesis in neuronal signaling.