Mitochondria capture and subsequently release Ca2+ ions, thereby sensing and shaping cellular Ca2+ signals. The Ca2+ uniporter MCU mediates Ca2+ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca2+ against Na+ or H +, respectively. Here we study the role of these ion exchangers in mitochondrial Ca2+ extrusion and in Ca2+-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca2+ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca2+ ([Ca2+]mt) elevations. NCLX overexpression enhanced the rates of Ca2+ efflux, whereas increasing LETM1 levels had no impact on Ca2+ extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca 2+]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na+/Ca2+ exchanger inhibitor CGP37157. The [Ca 2+]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na+/Ca2+ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca2+ extrusion from mitochondria. By controlling the duration of matrix Ca 2+ elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca2+ signals into redox changes.