Fatal arrhythmias are one of the main manifestations of ischemic heart disease in diabetic patients. Here, we investigated the effect of pretreatment with vildagliptin on myocardial arrhythmias, inflammatory responses, and expression of genes regulating mitochondrial biogenesis following cardiac ischemic injury in type II diabetic male Wistar rats. Chronic diabetes was modeled by a high-fat diet and low-dose streptozotocin method and lasted for 12 weeks. Vildagliptin (6 mg/dl) was orally administered during the last 4 weeks of the diabetic period. Then, rats' hearts (n = 8/each group) were immediately isolated and transferred to the Langendorff apparatus, in which left anterior descending coronary artery was tightened for 35 min to induce regional ischemia. Electrocardiography was continuously recorded and myocardial arrhythmias were interpreted according to the Lambeth Convention. Inflammatory cytokines in left ventricular samples were measured using ELISA kits, and gene expression was assayed using real-time PCR. Diabetic groups showed increased incidence and duration of ventricular fibrillation (VF) than controls (P < 0.05). Pretreatment of diabetic rats with vildagliptin resulted in a significant decrease in number, duration, and severity of premature ventricular complexes (PVC), tachycardia (VT), and VF during ischemia, compared to non-treated diabetic group (P < 0.05). Additionally, vildagliptin significantly increased the expression of genes PGC-1α, SIRT-1, and NRF-2 and reduced the levels of myeloperoxidase, creatine kinase release, and myocardial content of TNF-α and IL-1β in nondiabetic and diabetic rats as compared to corresponding controls (P < 0.01-0.05). Vildagliptin preconditioning reduced the occurrence and severity of fatal ventricular arrhythmias induced by myocardial ischemia in type II diabetic rats through increased activity of mitochondrial biogenesis-regulating genes and reduction of inflammatory reactions.