Sirtuin 4 (SIRT4) is a NAD+-dependent deacylase which suppresses fatty acid oxidation (FAO) and increases lipogenesis in muscle and white adipose tissue, whereas suppression of SIRT4 results in opposite effects. Since increased myocardial utilization and storage of lipids is a hallmark of diabetic heart disease, we aimed to evaluate the role of SIRT4 in the regulation of myocardial lipid metabolism in the normal and diabetic heart. Expression of SIRT4 was markedly reduced in human LV myocardium of diabetic individuals (-64%;p<0.05) compared to non-diabetic individuals. To investigate effects of reduced SIRT4 expression, 4 week-old mice with global deletion of SIRT4 (SIRT4-/-) were either treated with streptozotocin (60mg/d i.p.) to induce diabetes or with vehicle for 5 days and investigated after another 8 weeks. In isolated working hearts, FAO was increased (+46%;p<0.05) and glucose oxidation decreased (-41%;p<0.05) in vehicle-treated SIRT4-/- mice, accompanied by a 49% decrease in myocardial triglyceride levels (p<0.05). STZ treatment induced an increase in serum fatty acid and triglyceride levels and resulted in a similar increase of FAO and decrease of glucose oxidation in WT and SIRT4-/- mice. However, while myocardial triglyceride levels remained unchanged in WT mice following STZ treatment, STZ induced a marked increase in triglyceride levels in SIRT4-/- mice (2.8-fold). Shot-gun lipidomics revealed that acyl-carnitines were increased in STZ-treated WT mice but not SIRT4-/- mice. In parallel, levels and activity of malonyl-CoA decarboxylase were increased in STZ-treated WT but not SIRT4-/- mice. Thus, SIRT4 deficiency increases myocardial FAO and may balance the fate of fatty acids in the diabetic heart. These findings suggest that decreased SIRT4 activity in human diabetic hearts may causally contribute to increased FAO and lipid storage.