The balance between mitochondrial calcium (_mCa²⁺) uptake and efflux regulates ATP production, but if perturbed causes energy starvation or _mCa²⁺ overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of _mCa²⁺ efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic _mCa²⁺ overload. However, the mechanisms that regulate NCLX activity remain largely unknown. We used proximity biotinylation proteomic screening to identify the NCLX interactome and define novel regulators of NCLX function. Here, we discover the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances sodium (Na⁺)-dependent _mCa²⁺ efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in _mCa²⁺ efflux. Further, loss-of-function studies show that TMEM65 is required for Na⁺-dependent _mCa²⁺ efflux. Co-fractionation and in silico structural modeling of TMEM65 and NCLX suggest these two proteins exist in a common macromolecular complex in which TMEM65 directly stimulates NCLX function. In line with these findings, knockdown of Tmem65 in mice promotes _mCa²⁺ overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate that TMEM65 deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca²⁺ stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent _mCa²⁺ efflux, causing pathogenic _mCa²⁺ overload, cell death and organ-level dysfunction, and that gain of TMEM65 function mitigates these effects. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent _mCa²⁺ efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control of _mCa²⁺ homeostasis.

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