The balance between mitochondrial calcium ((m)Ca(2+)) uptake and efflux is essential for ATP production and cellular homeostasis. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of (m)Ca(2+) efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic (m)Ca(2+) overload. However, the mechanisms that regulate NCLX activity are largely unknown. Using proximity biotinylation proteomic screening, we identify the mitochondrial inner membrane protein TMEM65 as an NCLX binding partner that enhances sodium (Na(+))-dependent (m)Ca(2+) efflux. Mechanistically, acute pharmacological NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in (m)Ca(2+) efflux, and loss-of-function studies show that TMEM65 is required for Na(+)-dependent (m)Ca(2+) efflux. In line with these findings, knockdown of Tmem65 in mice promotes (m)Ca(2+) overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent (m)Ca(2+) efflux, causing pathogenic (m)Ca(2+) overload, cell death, and organ-level dysfunction. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent (m)Ca(2+) efflux and suggest modulation of TMEM65 as a therapeutic strategy for a variety of diseases.