We reported previously that glycerol is a substrate for energy production in cardiomyocytes. Increasing glycerol availability results in an increased glycerol uptake and its involvement in complex lipid biosynthesis and energy production. This study evaluated the relationship between glycerol supply, energy demand and intermediary metabolism leading to energy production. The work was performed on isolated rat heart perfused in the working mode. Glycerol concentrations modelled the fasting (0.33 mM) and fed (3.33 mM) states, respectively. Cardiac energy demand was modelled by increasing heart rate, from 350 to 450 bpm. Increasing glycerol supply increased glycerol uptake from 1.4 (350 bpm) to 3.8 (450 bpm) and from 9.7 (350 bpm) to 34.2 (450 bpm) µmol glycerol/heart in 30 min at 0.33 and 3.33 mM glycerol, respectively. At low glycerol supply, increasing heart rate did not influence the complex lipid synthesis. Conversely, high glycerol concentration increased the complex lipids synthesis by 5 and 30 fold at 350 and 450 bpm, respectively. Increasing glycerol supply and heart rate significantly increased glycerol oxidation rate. Moreover increasing glycerol supply did not affect glucose oxidation but increased palmitate uptake and significantly decreased its -oxidation. Physiological concentrations of glycerol contribute to the cardiac intermediary metabolism, both for energy production and glycerolipid synthesis. Increasing energy demand enhances the requirement and use of glycerol. Glycerol contributes to the regulation of cardiac metabolism and energy balance, mainly by decreasing the contribution of fatty acid oxidation and may thus represent a new factor in cardiac protection through the reduction of oxygen demand.