Abstract Body (Do not enter title and authors here): Introduction: Mitochondrial calcium (_mCa²⁺) exchange regulates energy metabolism, but if perturbed causes _mCa²⁺ depletion and energy starvation or _mCa²⁺ overload and cell death. The mitochondrial sodium (Na⁺)-calcium exchanger, NCLX, is critical for _mCa²⁺ efflux in the heart, and animal models support NCLX as a therapeutic target to limit pathogenic _mCa²⁺ overload. However, the mechanisms that regulate NCLX activity are largely unknown, representing a key barrier to translation. Goal: We used proximity biotinylation screening to identify the NCLX interactome and define novel regulators of NCLX function. Hypothesis: Our screen identified the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances Na⁺-dependent _mCa²⁺ efflux. Therefore, we hypothesized that TMEM65 promotes _mCa²⁺ efflux through NCLX. Approach: We measured _mCa²⁺ exchange in AC16 cardiomyocytes with TMEM65 overexpression (OE) or CRISPR/Cas9 genetic disruption of TMEM65 to test its effect on _mCa²⁺ efflux, and used pharmacologic inhibition and genetic knockout to determine TMEM65’s functional dependence on NCLX. We evaluated TMEM65’s impact on murine cardiac function in vivo using an AAV9-shRNA knockdown strategy. Results: Deletion of TMEM65 attenuated Na⁺-dependent _mCa²⁺ efflux, while inhibition or deletion of NCLX ablated the increase in _mCa²⁺ efflux observed with TMEM65 OE. Proximity ligation assay revealed co-localization of TMEM65 and NCLX in intact cells. In silico molecular modeling and co-fractionation of NCLX and TMEM65 via size-exclusion chromatography support their existence in a common macromolecular complex. Mutagenesis studies identified residues N163/D167 as critical to TMEM65 function, suggesting their importance to its cooperation with NCLX. TMEM65 expression decreased in human and murine heart failure, and Tmem65 KD in mice promoted myocardial _mCa²⁺ overload and impaired cardiac function. Notably, TMEM65 OE mitigated necrotic cell death during cellular Ca²⁺-overload in vitro. Conclusions: Loss of TMEM65 function disrupts NCLX-dependent _mCa²⁺ efflux, causing pathogenic _mCa²⁺ overload, cell death and organ-level dysfunction, whereas gain of TMEM65 function can attenuate these effects. Our findings demonstrate the essential role of TMEM65 in maintaining _mCa²⁺ efflux and suggest modulation of TMEM65 as a novel therapeutic strategy to control _mCa²⁺ homeostasis in heart failure and other pathologies featuring dysregulated _mCa²⁺ exchange.