Diabetic cardiomyopathy has been documented as an underlying etiology of heart failure (HF) among diabetics. Although oxidative stress has been proposed to contribute to diabetic cardiomyopathy, much of the evidence lacks specificity. Furthermore, whether alterations occur at the cardiac proteome level in diabetic cardiac complications with attendant oxidative stress remains unknown. Therefore, we sought to identify cardiac protein changes in relation to myocardial oxidative stress that are specific to diabetic cardiomyopathy. Diabetes was induced in rats by a single injection of streptozotocin (STZ). STZ-treated rats were examined for diabetic cardiomyopathy at 8 weeks post-STZ by left ventricular (LV) hemodynamic analysis. LV systolic pressure (LVSP), rate of pressure rise (+dP/dt), and rate of pressure decay (-dP/dt) were depressed while LV end-diastolic pressure (LVEDP) was increased. Myocardial oxidative stress was increased in STZ-diabetic rats, as indexed by significant increases in myocardial formation of 8-iso PGF(2alpha) and oxidized glutathione (GSSG). In-depth mining of the diabetic myocardial proteome by proteomic analysis utilizing two-dimensional difference gel electrophoresis and mass spectrometry (DIGE/MS) techniques revealed that a high proportion (12 of 24) of the altered proteins that could be identified by mass spectrometry were localized to the mitochondria. Down-regulation of antioxidant and anti-apoptotic proteins was also observed in STZ-diabetic hearts. These results characterize a specific 'type I diabetic' pattern of cardiac proteome changes indicative of diabetic cardiomyopathy presenting with higher oxidative stress, supporting the idea that analysis of isoprostane biosynthesis and protein expression profiles may be useful diagnostically to assess the efficacy of antioxidant therapies as prophylactic treatments against type I diabetes mellitus complications involving the heart.