Abstract Mitochondrial calcium (Ca 2+ mito ) dynamics plays vital roles in regulating fundamental cellular and organellar functions including bioenergetics. However, neuronal Ca 2+ mito dynamics in vivo and its regulation by brain activity are largely unknown. By performing two-photon Ca 2+ imaging in the primary motor (M1) and visual cortexes (V1) of awake behaving mice, we find that discrete Ca 2+ mito transients occur synchronously over somatic and dendritic mitochondrial network, and couple with cytosolic calcium (Ca 2+ cyto ) transients in a probabilistic, rather than deterministic manner. The amplitude, duration, and frequency of Ca 2+ cyto transients constitute important determinants of the coupling, and the coupling fidelity is greatly increased during treadmill running (in M1 neurons) and visual stimulation (in V1 neurons). Moreover, Ca 2+ /calmodulin kinase II is mechanistically involved in modulating the dynamic coupling process. Thus, activity-dependent dynamic Ca 2+ mito -to-Ca 2+ cyto coupling affords an important mechanism whereby Ca 2+ mito decodes brain activity for the regulation of mitochondrial bioenergetics to meet fluctuating neuronal energy demands as well as for neuronal information processing.