Introduction: The metabolic modulator trimetazidine (TMZ) has been suggested to induce a metabolic shift from myocardial fatty acid oxidation (FAO) to glucose utilization, but this mechanism remains unproven in humans. The oxidation of plasma derived FA is commonly measured in humans, whereas the contribution of FA from triglycerides stored in the myocardium has been poorly characterized. Aims: To verify the hypothesis that TMZ induces a metabolic shift, we combined positron emission tomography (PET) and magnetic resonance spectroscopy (1H-MRS) to measure myocardial FAO from plasma and intracellular lipids, and myocardial glucose metabolism. Nine obese subjects were studied before and after 1 month of TMZ treatment. Myocardial glucose and FA metabolism were assessed by PET with 18F-fluorodeoxyglucose and 11C-palmitate. 1H-MRS was used to measure myocardial lipids, the latter being integrated into the PET data analysis to quantify myocardial triglyceride turnover. Results: Myocardial FAO derived from intracellular lipids was at least equal to that of plasma FAs (P= NS). BMI and cardiac work were positively associated with the oxidation of plasma derived FA (P≤ 0.01). TMZ halved total and triglyceride-derived myocardial FAO (32.7 ± 8.0 to 19.6 ± 4.0 μmol/min and 23.7 ± 7.5 to 10.3 ± 2.7 μmol/min, respectively; P≤ 0.05). These changes were accompanied by increased cardiac efficiency since unchanged LV work (1.6 ± 0.2 to 1.6 ± 0.1 Watt/g × 102, NS) was associated with decreased work energy from the intramyocardial triglyceride oxidation (1.6 ± 0.5 to 0.4 ± 0.1 Watt/g × 102, P= 0.036). Conclusions: In obese subjects, we demonstrate that myocardial intracellular triglyceride oxidation significantly provides FA-derived energy for mechanical work. TMZ reduced the oxidation of triglyceride-derived myocardial FAs improving myocardial efficiency.