The versatility and universality of Ca2+ as intracellular messenger is guaranteed by the compartmentalization of changes in [Ca2+]. In this context, mitochondrial Ca2+ plays a central role, by regulating both specific organelle functions and global cellular events. This versatility is also guaranteed by a cell type-specific Ca2+ signaling toolkit controlling specific cellular functions. Accordingly, mitochondrial Ca2+ uptake is mediated by a multimolecular structure, the MCU complex, which differs among various tissues. Its activity is indeed controlled by different components that cooperate to modulate specific channeling properties. We here investigate the role of MICU3, an EF-hand containing protein expressed at high levels, especially in brain. We show that MICU3 forms a disulfide bond-mediated dimer with MICU1, but not with MICU2, and it acts as enhancer of MCU-dependent mitochondrial Ca2+ uptake. Silencing of MICU3 in primary cortical neurons impairs Ca2+ signals elicited by synaptic activity, thus suggesting a specific role in regulating neuronal function.