Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2 (Argonaute2), a core member of miRNA machinery, remain elusive.We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes and diabetic cardiomyopathy. Recombinant adeno-associated virus type 9 was used to deliver Ago2 to mice through the tail vein. Cardiac structure and functions were assessed by echocardiography and catheter manometer system.Ago2 was decreased in mitochondria of diabetic cardiomyocytes. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain subunits and decrease reactive oxygen species. Malonylation, a posttranslational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function.Our findings reveal that the SIRT3-Ago2-CYTB axis links glucotoxicity to cardiac electron transport chain imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.