Mitochondria not only provide cells with energy, but are central to Ca2+signaling. Powered by the mitochondrial membrane potential, Ca2+enters the mitochondria and is released into the cytosol through a mitochondrial Na+/Ca2+exchanger. Weestablished that NCLX, a newly discovered mitochondrial Na+/Ca2+exchanger, is expressed in astrocytes isolated from mice of either sex. Immunoblot analysis of organellar fractions showed that the location of NCLX is confined to mitochondria. Using pericam-based mitochondrial Ca2+imaging and NCLX inhibition either by siRNA or by the pharmacological blocker CGP37157, we demonstrated that NCLX is responsible for mitochondrial Ca2+ extrusion. Suppression of NCLX function altered cytosolic Ca2+ dynamics in astrocytes and this was mediated by a strong effect of NCLX activity on Ca2+ influx via store-operated entry. Furthermore, Ca2+ influx through the store-operated Ca2+ entry triggered strong, whereas ER Ca2+release triggered only modest mitochondrial Ca2+transients, indicating that the functional cross talk between the plasma membrane and mitochondrial domains is particularly strong in astrocytes. Finally, silencing of NCLX expression significantly reduced Ca2+-dependent processes in astrocytes (i.e., exocytotic glutamate release, in vitro wound closure, and proliferation), whereas Ca2+ wave propagation was not affected. Therefore, NCLX, by meditating astrocytic mitochondrial Na+/Ca2+ exchange, links between mitochondria and plasma membrane Ca2+signaling, thereby modulating cytoplasmic Ca2+transients required to control a diverse array of astrocyte functions. © 2013 the authors.