In physiological conditions, the energy required for cardiac contraction is provided by the β-oxidation of long chain free fatty acids (i.e. palmitate) in the mitochondrial matrix. To diffuse through the mitochondrial membrane, palmitate formed a complex with carnitine called palmitoyl-carnitine (PC). In order to reach the contractile demand, the rate of ATP synthesis is dynamically regulated through a mechanism called excitation-metabolism coupling (EMC). The EMC depends on the Ca2+ movement between the mitochondria and the sarcoplasmic reticulum (SR). In obesity and type 2 diabetes, patients develops a diabetic cardiomyopathy (DC), characterized by a ventricular dysfunction, with contractile and Ca2+ signaling disturbances, associated with a mitochondrial dysfunction. In the DC, the increase of fatty acid oxidation is associated with a decrease of ATP/Oxygen ratio, indicating a mitochondrial uncoupling. Although long chain fatty acid also alter Ca2+ handling, the cross regulation between mitochondria uncoupling and Ca2+ handling are not fully understood. In this work we aimed to study the effect of an acute application of PC on Ca2+ signaling and mitochondrial function on left ventricles of control C57/BL6 mice. The application of 10µM of PC on isolated mitochondria decreases the Adenine Nucleotide Transporter (ANT) activity and induced a depolarization of mitochondrial membrane. This depolarization is also observed in freshly isolated cardiomyocytes and is associated with a massive increase of mitochondrial ROS production. This PC-induced ROS production increases diastolic SR Ca2+ leak through the oxidation of SR Ca2+ release channels (the ryanodine receptor 2 (RyR2)). This disturbance of Ca2+ handling is associated to an increase of arrhythmia episodic. Inhibition of ANT using bongrekic acid prevents the mitochondrial defects, the RyR2 oxidation, altered Ca2+ handling and arrhythmia leading by an acute application of PC. Altogether, these results suggest that an acute elevation of long chain fatty acids disturbed ANT activity which affects Ca2+ handling. Disturbance of ANT activity and increase ROS production may thus contributes to development of DC.