Cardiac complications and heart failure are the leading cause of death in type 2 diabetic patients. Mitochondrial dysfunction is central in the pathogenesis of the type 2 diabetic heart. However, it is unclear whether this dysfunction is specific for a particular subcellular region. The purpose of this study was to determine whether mitochondrial dysfunction in the type 2 diabetic heart is specific to a spatially distinct subset of mitochondria. We investigated mitochondrial morphology, function, and proteomic composition of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) in 18-wk-old db/db mice. Oxidative damage was assessed in subpopulations through the measurement of lipid peroxidation byproducts and nitrotyrosine residues. Proteomic profiles and posttranslational modifications were assessed in mitochondrial subpopulations using iTRAQ and multi-dimensional protein identification technologies, respectively. SSM from db/db hearts had altered morphology, including a decrease in size and internal complexity, whereas db/db IFM were increased in internal complexity. Db/db SSM displayed decreased state 3 respiration rates, electron transport chain activities, ATP synthase activities, and mitochondrial membrane potential and increased oxidative damage, with no change in IFM. Proteomic assessment revealed a greater impact on db/db SSM compared with db/db IFM. Inner mitochondrial membrane proteins, including electron transport chain, ATP synthesis, and mitochondrial protein import machinery, were predominantly decreased. We provide evidence that mitochondrial dysfunction in the type 2 diabetic heart is associated with a specific subcellular locale. Furthermore, mitochondrial morphological and functional indexes are impacted differently during type 2 diabetic insult and may result from the modulation of spatially distinct mitochondrial proteomes.