Mitochondrial ability of shaping Ca²⁺ signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca²⁺ uniporter (MCU) and of unique targeted Ca²⁺ probes to directly address this issue. We demonstrate that, during spontaneous Ca²⁺ pacing, Ca²⁺ peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca²⁺ hot spots generated on the OMM surface. Cytoplasmic Ca²⁺ peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca²⁺ rises.