Background: Iron in the mitochondria is regulated to maintain mitochondrial physiological function. A loss of mitoNEET protein located in the outer mitochondrial membrane has been shown to increase mitochondrial iron content in adipocytes. However, the role of mitoNEET in iron homeostasis has not been determined in the heart. Methods and Results: MitoNEET flox/flox (mNT f/f ) mice were generated with lox-P and homologous recombination strategies. Cardiac-specific deletion of mitoNEET was achieved using αMHC-Cre (mNT -/- ). Mice, mNT -/- (n=11) and mNT f/f (n=10), were bred for 3 months. Mitochondrial iron content, measured by colorimetric method, was significantly increased in mNT -/- mice compared to mNT f/f mice (1.13±0.09 vs. 0.58±0.07 μg/dl/μg mitochondrial protein, P<0.001). In parallel, immunoblot analysis showed that mitochondrial ferritin, mitochondrial iron transporter, was significantly higher in mNT -/- than mNT f/f by 45%. Mitoferrin2, mitochondrial iron importer, and ATP-binding cassette transporter type B8, mitochondrial iron exporter, protein did not differ between groups. The activity of electron transport chain complex V, mitochondrial iron importer, was also higher in mNT -/- than mNT f/f (617±22 vs. 496±17 mM/min/mg mitochondrial protein, P<0.05). In contrast, frataxin, involved in the synthesis of iron-sulfur cluster (ISC), was significantly lower in mNT -/- by 49%. ATP-binding cassette transporter type B7, mitochondrial ISC exporter, did not differ. Body weight and heart weight were comparable between groups. Left ventricular end-diastolic diameter (2.9±0.1 vs. 2.9±0.1 mm), percent fractional shortening (55±3 vs. 55±3 %), and wall thickness (0.71±0.01 vs. 0.73±0.01 mm) measured by echocardiography were also similar. Conclusions: Cardiac specific deletion of mitoNEET protein increased mitochondrial iron content in mice, in association with the increase in iron importer and transporter and the decrease in frataxin, suggesting that mitoNEET may play an important role in the regulation of mitochondrial function via iron homeostasis in the heart.