Abstract Body: Introduction: Autophagy-related protein 7 (Atg7) is a key regulator of macroautophagy; however, its role in selective mitochondrial autophagy (mitophagy) in the heart remains incompletely understood. We used complementary biochemical and genetic approaches to investigate whether Atg7 regulates mitophagy in cardiomyocytes and intact hearts. Objectives: To define the molecular mechanisms by which Atg7 promotes mitophagy in cardiomyocytes and the heart using complementary biochemical and genetic approaches. Methods and Results: Subcellular fractionation and quantum dot–labeled electron microscopy revealed that Atg7 localizes to cardiac mitochondria. These findings were further confirmed in isolated mitochondrial preparations, where Atg7 co-localized with Mitotracker-labeled mitochondria. To determine whether Atg7 promotes mitophagy, primary cardiomyocytes were transduced to overexpress Atg7 and treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce mitochondrial stress. Mitophagy was quantified using a pH-sensitive fluorescent mitophagy reporter dye. Atg7 overexpression significantly increased the number of red mitophagy puncta compared with β-gal–expressing control cardiomyocytes. To examine mitophagy activation in vivo, cardiac-specific Atg7 transgenic mice (αMHC-Atg7 Tg) and littermate non-transgenic controls were treated with CCCP. Hearts from αMHC-Atg7 Tg mice displayed increased accumulation of the autophagolysosomal marker LC3-II in mitochondrial fractions, suggesting enhanced mitochondrial autophagic processing. To directly measure mitophagy flux in vivo, αMHC-Atg7 Tg mice were crossed with mitochondrial-targeted Keima (Mt-Keima) reporter mice. Hearts from αMHC-Atg7 x Mt-Keima mice exhibited significantly greater red-to-green fluorescence ratios, indicating increased delivery of mitochondria to lysosomes. Conclusions: Our research demonstrates that Atg7 localizes to cardiac mitochondria and promotes recruitment of mitophagy machinery, thereby enhancing basal mitophagy flux in the heart. These results identify Atg7 as a critical regulator of mitochondrial quality control in cardiomyocytes.