Objectives: To assess the effect of vitamin D-2 and to elucidate the underlying mechanisms on acute myocardial injury induced by isoproterenol (ISO) in diabetic rats. Methods: Rats were divided into control rats, diabetic rats (DM), diabetic rats received ISO (DM-ISO), and diabetic rats pretreated with vitamin D-2 and received ISO (DM-D-2-ISO). Results: Vitamin D-2 pretreatment significantly decreased fasting glucose and myocardial malondialdehyde, associated with increased insulin, myocardial glutathione and superoxide dismutase in DM-D-2-ISO versus DM-ISO. The serum triglycerides, total cholesterol, and LDL were significantly decreased, along with increased HDL and adiponectin. Poly-ADP ribose polymerase, cyclooxygenase-2, tumour necrosis factor alpha, interleukin-6, caspase-3, BAX, and p53 were significantly downregulated in myocardium of DM-D-2-ISO versus DM-ISO. Histological studies showed diminished inflammatory cells infiltration in myocardium of DM-D-2-ISO versus DM-ISO. Conclusion: Vitamin D-2 ameliorates hyperglycaemia, dyslipidaemia, redox imbalance, inflammatory and apoptotic processes, protecting the myocardium of diabetic rats against acute myocardial infarction.