When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca²⁺ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca²⁺ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca²⁺ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca²⁺/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na⁺ or Ca²⁺ extrusion, and in mouse dentate granule cells, Ca²⁺ creates the majority of this demand.