Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1 h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.