A proposed mechanism of neuronal death associated with a variety of neurodegenerative diseases is the response of neurons to oxidative stress and consequent cytosolic Ca²⁺ overload. One hypothesis is that cytosolic Ca²⁺ overload leads to mitochondrial Ca²⁺ overload and prolonged opening of the permeability transition pore (PTP), resulting in mitochondrial dysfunction. Elimination of cyclophilin D (CyPD), a key regulator of the PTP, results in neuroprotection in a number of murine models of neurodegeneration in which oxidative stress and high cytosolic Ca²⁺ have been implicated. However, the effects of oxidative stress on the interplay between cytosolic and mitochondrial Ca²⁺ in adult neurons and the role of the CyPD‐dependent PTP in these dynamic processes have not been examined. Here, using primary cultured cerebral cortical neurons from adult wild‐type (WT) mice and mice missing cyclophilin D (CyPD‐KO), we directly assess cytosolic and mitochondrial Ca²⁺, as well as ATP levels, during oxidative stress. Our data demonstrate that during acute oxidative stress mitochondria contribute to neuronal Ca²⁺ overload by release of their Ca²⁺ stores. This result contrasts with the prevailing view of mitochondria as a buffer of cytosolic Ca²⁺ under stress conditions. In addition, we show that CyPD deficiency reverses the release of mitochondrial Ca²⁺, leading to lower of cytosolic Ca²⁺ levels, attenuation of the decrease in cytosolic and mitochondrial ATP, and a significantly higher viability of adult CyPD‐knockout neurons following exposure of neurons oxidative stress. The study offers a first insight into the mechanism underlying CyPD‐dependent neuroprotection during oxidative stress.