The execution of proper Ca²⁺ signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca²⁺ released from the ER is “quasi-synaptically” transferred to mitochondrial matrix, where Ca²⁺ stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca²⁺ transfer is excessive and sustained, mitochondrial Ca²⁺ overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca²⁺ transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca²⁺ flux and describe the activities of the various Ca²⁺ players based on their primary intra-organelle localization.