Ca²⁺/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a key regulator of neuronal signaling and synaptic plasticity. Synaptic activity and neurotransmitter homeostasis are closely coupled to the energy metabolism of both neurons and astrocytes. However, whether CaMKIIα function is implicated in brain energy and neurotransmitter metabolism remains unclear. Here, we explored the metabolic consequences of CaMKIIα deletion in the cerebral cortex using a genetic CaMKIIα knockout (KO) mouse. Energy and neurotransmitter metabolism was functionally investigated in acutely isolated cerebral cortical slices using stable ¹³C isotope tracing, whereas the metabolic function of synaptosomes was assessed by the rates of glycolytic activity and mitochondrial respiration. The oxidative metabolism of [U-¹³C]glucose was extensively reduced in cerebral cortical slices of the CaMKIIα KO mice. In contrast, metabolism of [1,2-¹³C]acetate, primarily reflecting astrocyte metabolism, was unaffected. Cellular uptake, and subsequent metabolism, of [U-¹³C]glutamate was decreased in cerebral cortical slices of CaMKIIα KO mice, whereas uptake and metabolism of [U-¹³C]GABA were unaffected, suggesting selective metabolic impairments of the excitatory system. Synaptic metabolic function was maintained during resting conditions in isolated synaptosomes from CaMKIIα KO mice, but both the glycolytic and mitochondrial capacities became insufficient when the synaptosomes were metabolically challenged. Collectively, this study shows that global deletion of CaMKIIα significantly impairs cellular energy and neurotransmitter metabolism, particularly of neurons, suggesting a metabolic role of CaMKIIα signaling in the brain. (Figure presented.).