Introduction: The balance between mitochondrial calcium (mCa) uptake and efflux regulates ATP production, but if perturbed causes mCa depletion and energy starvation or mCa overload and cell death. Disrupted mCa homeostasis is implicated in ischemia-reperfusion injury, heart failure, and neurodegeneration. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of mCa efflux in excitable tissues, supporting NCLX as a promising therapeutic target to limit pathogenic mCa overload in organs like the heart and brain. However, a critical barrier to translation is that little is known about the mechanisms controlling NCLX activity.Goals: We used proximity biotinylation screening to identify the NCLX interactome and proteins that may regulate NCLX function. We identified TMEM65, a mitochondrial inner membrane protein of unknown function, as an NCLX-proximal protein.Hypothesis: Null mutation of TMEM65 causes mitochondrial encephalomyopathy and phenocopies genetic NCLX disruption. Therefore, we hypothesized that TMEM65 modulates mCa efflux through NCLX. Approach: Here, we used measurements of mCa exchange in AC16 cardiomyocytes with TMEM65 overexpression (OE) or CRISPR/Cas9 genetic disruption of TMEM65 to test TMEM65’s effect on mCa flux and its functional dependence on NCLX. We used AAV-mediated Tmem65 knockdown (KD) and TMEM65 OE in mice to assess the effects of gain- or loss of TMEM65 expression on cardiac function.Results: TMEM65 OE potently enhanced mCa efflux in vitro, and NCLX inhibition with CGP-37157 mitigated this effect. Knockout of TMEM65 attenuated mCa efflux. In vivo Tmem65 KD reduced left ventricular fractional shortening and reduced S293 phosphorylation of pyruvate dehydrogenase in the heart, indicative of increased mCa loading. In vivo TMEM65 OE did not alter cardiac function.Conclusions: We conclude that TMEM65 has an essential function to promote mCa efflux, likely via positive regulation of NCLX, and is critical to limiting deleterious mCa overload in the intact heart. Ongoing work is defining the functional interactions between TMEM65 and NCLX, and establishing the utility of targeting TMEM65 to restore mCa homeostasis in heart failure and other excitable tissue diseases.