Abstract Hyperlipidemia represents a major risk factor for cardiovascular diseases leading to myocardial injury. The present study aimed to illustrate the myocardial injury induced in a diabetic hyperlipidemic rat model and the effect of vitamin D3, 10-DHGD intake either individually or in combination form. Male albino rats were selected for the study, received alloxan, hypercholesterolemic diet, and categorized into four groups. The first one (DHC), received hypercholesterolemic diet only and referred to as control. The remaining groups (2, 3, 4) received vitamin D3, 10-DHGD, and combination of both, respectively. Certain biomarkers that were selected for MI evaluation included blood glucose, lipogram pattern, Copeptin, C-reactive protein, myeloperoxidase, heart fatty acid-binding protein, and histopathological changes in myocardium and aorta. Vitamin D3 and 10-DHGD intake induced significant hypoglycemic, hypolipidemic effects, decreased inflammation, and MI biomarkers. Decreased myocardial vacuoles, foam cells, and intimal lesions were also observed compared to DHC. Their combination intake induced more marked reduction in all biomarkers and showed a histopathological pattern similar to normal features of myocardium and aorta. Our findings suggest the therapeutic roles of vitamin D3, 10-DHGD, and their combination against myocardial injury in diabetic hyperlipidemic rats.