Diabetic patients are prone to metabolic perturbations that progressively contribute to structural, functional and proteomic alterations in the myocardium. Phosphodiesterase-5 (PDE-5) inhibitors exhibit cardioprotective effects against ischemic/reperfusion injury, however the effects of chronic administration of PDE-5 inhibitors, particularly under diabetic conditions, remain unknown. Hence, the present study was designed to identify novel protein targets related to long-acting PDE-5 inhibitor tadalafil-induced cardioprotection in diabetes. Using two-dimensional differential in-gel electrophoresis with 3 CyDye labeling and MALDI–TOF/TOF tandem mass spectrometry we identified alterations in the expressions of cardiac proteins in diabetic db/db mice treated with tadalafil. Tadalafil reversed the coordinated alterations of cytoskeletal/contractile proteins such as myosin light chain (MLY) 2 and 4, myosin heavy chain α and myosin-binding protein C which contributes to contractile dysfunction. The expression of intermediate filament protein vimentin and extra-cellular matrix proteins like cysteine and glycine rich protein-3 and collagen type VI α were upregulated in db/db mice indicating cardiac remodeling in diabetes. These detrimental proteomic alterations were reflected in cardiac function which were reversed in tadalafil treated mice. Tadalafil also enhanced antioxidant enzyme glutathione S-transferase Kappa-1 (GSKT-1) and downregulated redox regulatory chaperones like heat shock protein 8 (HSPA8), and 75 kD glucose regulatory protein (75GRP). Furthermore, tadalafil treatment significantly attenuated GSSG/GSH ratio and improved the metabolic status of db/db mice. Chronic treatment with tadalafil in db/db mice modulates proteins involved in cytoskeletal rearrangement and redox signaling of the heart, which may explain the beneficial effects of PDE-5 inhibition in diabetes.