Background: Mitochondria are the powerhouses of the cell, supplying a continuous supply of energy in the form of ATP. Maintenance of mitochondrial structure and function is governed by two opposing, dynamic processes known as fusion and fission. These protect the integrity of the mitochondrial genome as well as removing unhealthy mitochondria via PINK1-Parkin-mediated mitophagy. However, mitochondrial dysfunction is closely associated with diabetic cardiomyopathy. Interestingly, the fusion protein mitofusin-2 (Mfn2) has been implicated in the pathogenesis of diabetes. Nonetheless, the role of Mfn2 in the heart is poorly characterized. Therefore, the aim of this study was to investigate changes to cardiac mitochondrial structure and function in a diabetic model, with a particular focus upon fusion/fission. Methods: Diabetes was induced in male Wistar rats via a single intraperitoneal injection of streptozotocin (55 mg/kg in 0.9% w/v saline) then terminally anaesthetised 16 weeks post-injection. All procedures were performed in accordance with Scientific Procedures Act (1986). Left ventricle tissue was isolated and changes to protein expression assessed using qPCR, Western blot and quantitative mass spectrometry (MS). Mitochondrial OXPHOS function was investigated using enzyme activity assays. Data was expressed as mean±SEM and analysed by t-test or ANOVA. P values <0.05 were deemed significant. Results: STZ-treated rats developed significant hyperglycaemia 16 weeks post-injection (p<0.0001, n=6). Western blot showed a significant increase in Mfn2 expression levels in STZ compared to controls (p=0.0386, n=6) with a similar fold change shown in MS (1.3 fold increase, p=0.0035, n=6). In comparison, there was a downwards trend in PINK1 expression at the transcript and protein level (p=0.0791 and p=0.1271 respectively, n=6). Functional studies of isolated mitochondria revealed a significant decrease in activity of complex I, IV and V (p=0.0095, p=0.0190 and p=0.0254, n=6 respectively). Quantitative MS identified 1437 protein and analysis using Ingenuity Pathway Analysis® software highlighted an increase in mitochondrial dysfunction and β oxidation which corroborates the functional assays. Conclusion: Upregulation of Mfn2 is associated with mitochondrial dysfunction in the diabetic myocardium. Increased Mfn2 levels may be a result of an imbalance in mitochondrial dynamics. Future work aims to investigate Mfn2 regulation in the diabetic heart by using cell culture, as well as a focus on whether changes to mitochondrial function are linked to changes in structure using electron microscopy. These studies will provide invaluable information as to the role Mfn2 plays in the pathogenesis of cardiac mitochondrial dysfunction in diabetes with the hope to identify potential future targets for therapeutic intervention.