Diabetic cardiomyopathy (DCM) is initiated independently of other cardiac risk factors and is characterized by an early, often clinically silent decline in myocardial function. Nucleoside-diphosphate-kinase B (NDPK-B) plays a critical role in ion channel regulation and signal transduction, and its genetic deletion in middle-aged mice induces a phenotype resembling diabetic cardiomyopathy. The present study investigated the contribution of protein O-GlcNAcylation, driven by the hexosamine biosynthesis pathway (HBP), to the initiation and progression of DCM. NDPK-B-deficient prediabetic mice and streptozotocin-induced diabetic mice were examined. In both models, left ventricles (LV) exhibited increased extracellular matrix deposition and a marked diastolic dysfunction. Concomitantly, activation of the HBP was observed in prediabetic and diabetic LVs. Proteomic profiling of O-GlcNAcylated proteins revealed substantial dysregulation of mitochondrial proteins under prediabetic conditions, particularly affecting components of respiratory complex I, along with metabolic fuel reprogramming and activation of immune and inflammatory pathways. Progression to overt DCM was associated with a shift toward lipid-driven metabolic remodeling and profound structural mitochondrial impairment. These findings identify aberrant mitochondrial O-GlcNAcylation as a key early event in the initial stages of cardiac damage and reveal a mechanism involved in prediabetic cardiomyopathy.