Abstract Body: Introduction: Increased mitochondrial Ca²⁺ (_mCa²⁺) content matches ATP production to cellular demand, but in excess triggers cell death. We recently found that the mitochondrial inner membrane protein TMEM65 interacts with and promotes _mCa²⁺ efflux via the mitochondrial Na⁺/Ca²⁺ exchanger, NCLX. Deletion of TMEM65 in vitro increases _mCa²⁺ accumulation, suggesting that TMEM65 is critical for _mCa²⁺ homeostasis. TMEM65 transcripts are downregulated in failing hearts, but TMEM65's functional relevance in the healthy heart, and how altered TMEM65 expression may influence heart failure progression, is unresolved. Hypothesis: We hypothesized that loss of TMEM65 contributes to _mCa²⁺ overload, leading to cardiomyocyte dropout and contractile dysfunction. Goals: We generated mice with tamoxifen-inducible, cardiomyocyte-specific Tmem65 knockout (αMHC-MCM x Tmem65^fl/fl) to assess the primary effects of loss of cardiomyocyte TMEM65 expression on adult heart function. We also generated mice (αMHC-MCM x Tmem65^fl/fl x Mcu^fl/fl) with simultaneous knockout of Mcu, the mitochondrial calcium uniporter, to distinguish _mCa²⁺-dependent vs. _mCa²⁺-independent effects of Tmem65 deletion. Approach: Adult mice were subjected to echocardiography to assess baseline cardiac function, then administered tamoxifen chow for 3 weeks. Echocardiography was repeated after 5 weeks of tamoxifen washout. Results: Western blotting confirmed loss of TMEM65 protein from cardiomyocytes of tamoxifen-treated αMHC-MCM x Tmem65^fl/fl mice. Loss of cardiomyocyte TMEM65 significantly reduced left ventricular fractional shortening within 8 weeks of gene deletion. We also observed a trend towards downregulation of proteins involved in _mCa²⁺ uptake in TMEM65-null cardiomyocytes, hinting at compensatory adaptation that could limit _mCa²⁺ overload and further functional decline. Indeed, simultaneous deletion of Mcu rescued the contractile deficit observed with deletion of Tmem65 alone. Conclusions: Our findings indicate that acute loss of TMEM65 impairs the contractile performance of the adult heart, likely by predisposing cardiomyocytes to deleterious _mCa²⁺ overload. Ongoing studies seek to evaluate the therapeutic potential of targeting TMEM65 to improve _mCa²⁺ handling and cardiac function in heart failure.