Mitochondrial calcium (sub.mCasup.2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Nasup.+/Casup.2+ exchanger (NCLX), which is proposed to be the primary mechanism for sub.mCasup.2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to sub.mCasup.2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting sub.mCasup.2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of sub.mCasup.2+ efflux in cellular function and suggest that augmenting sub.mCasup.2+ efflux may be a viable therapeutic strategy in disease.