Mitochondrial Ca²⁺ homeostasis-composed of the balance of Ca²⁺ influx and efflux-is responsible for the control of numerous cellular functions, including energy metabolism, the generation of reactive oxygen species, the spatiotemporal dynamics of Ca²⁺ signaling, as well as cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca²⁺ uniporter (MCU) provides new possibilities for applying genetic approaches to study the mitochondrial Ca²⁺ influx mechanism in various cell types and tissues. In addition, the successive discovery of various auxiliary subunits associated with the MCU suggests that mitochondrial Ca²⁺ uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex referred to as MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU post-translational modifications in the regulation of mitochondrial Ca²⁺ uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane, which could significantly regulate mitochondrial Ca²⁺ handling as well as numerous mitochondrial and cellular functions. In this review, we discuss the current literature on mitochondrial Ca²⁺ uptake mechanisms, with a particular focus on the structure and function of mtCUC as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.