ABSTRACTMitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+oscillations by increasing the light frequency, intensity and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1, encoded byDNM1L), via the activation of Ca2+-dependent kinases CaMKII, ERK and CDK1. However, optogenetically engineered Ca2+signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins mitofusin 1 (MFN1) and 2 (MFN2). Overall, this study provides an effective and innovative approach to altering Ca2+signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.