Mitochondrial Ca2+ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics. Recently developed GCaMP genetically-encoded Ca2+ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca2+ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca2+ dynamics in culture, revealing Ca2+ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca2+ indicators, our results show that mitochondrial Ca2+ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca2+ dynamics on cytosolic Ca2+ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca2+ increase using mito-GCaMP5G and a synthetic organic Ca2+ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca2+ signaling pathway, our results demonstrated that the mitochondrial Ca2+ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.