RATIONALE: Diabetic patients develop cardiomyopathy characterized by hypertrophy, diastolic dysfunction, and intracellular lipid accumulation, termed lipotoxicity. Diabetic hearts utilize fatty acids as a major energy source, which produces high levels of oxidative stress, thereby inducing mitochondrial dysfunction. OBJECTIVE: To elucidate how mitochondrial function is regulated in diabetic cardiomyopathy. METHODS AND RESULTS: Mice were fed either a normal diet or high-fat diet (HFD, 60 kcal % fat). Although autophagic flux was activated by HFD consumption, peaking at 6 weeks ( P<0.05), it was attenuated thereafter. Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD feeding (mitophagy area: 8.3% per cell with normal diet and 12.4% with HFD) and continued to increase even after 2 months ( P<0.05). By isolating adult cardiomyocytes from GFP-LC3 mice fed HFD, we confirmed that mitochondria were sequestrated by LC3-positive autophagosomes during mitophagy. In wild-type mice, cardiac hypertrophy, diastolic dysfunction (end diastolic pressure-volume relationship =0.051±0.009 in normal diet and 0.11±0.004 in HFD) and lipid accumulation occurred within 2 months of HFD feeding ( P<0.05). Deletion of atg7 impaired mitophagy, increased lipid accumulation, exacerbated diastolic dysfunction (end diastolic pressure-volume relationship =0.11±0.004 in wild type and 0.152±0.019 in atg7 cKO; P<0.05) and induced systolic dysfunction (end systolic pressure-volume relationship =24.86±2.46 in wild type and 15.93±1.76 in atg7 cKO; P<0.05) during HFD feeding. Deletion of Parkin partially inhibited mitophagy, increased lipid accumulation and exacerbated diastolic dysfunction (end diastolic pressure-volume relationship =0.124±0.005 in wild type and 0.176±0.018 in Parkin KO, P<0.05) in response to HFD feeding. Injection of TB1 (Tat-Beclin1) activated mitophagy, attenuated mitochondrial dysfunction, decreased lipid accumulation, and protected against cardiac diastolic dysfunction (end diastolic pressure-volume relationship =0.110±0.009 in Control peptide and 0.078±0.015 in TB1, P<0.05) during HFD feeding. CONCLUSIONS: Mitophagy serves as an essential quality control mechanism for mitochondria in the heart during HFD consumption. Impairment of mitophagy induces mitochondrial dysfunction and lipid accumulation, thereby exacerbating diabetic cardiomyopathy. Conversely, activation of mitophagy protects against HFD-induced diabetic cardiomyopathy.