Patients with mutations in the mitochondrial very-long-chain Acyl-CoA dehydrogenase (VLCAD) gene are at risk for cardiomyopathy, myocardial dysfunction, ventricular tachycardia (VT), and sudden cardiac death. The mechanism is not known. Here we report a novel mechanism of VT in mice lacking VLCAD (VLCAD-/-). These mice exhibited polymorphic VT and increased incidence of VT after isoproterenol infusion. Polymorphic VT was induced in 10 out of 12 VLCAD-/- mice (83%) when isoproterenol was used. One out of 10 VLCAD deficient mice with polymorphic VT had VT with the typical bidirectional morphology. At the molecular level, VLCAD deficient cardiomyocytes showed increased levels of cardiac ryanodine receptor 2, phospholamban and calsequestrin with increased [3H] ryanodine binding in heart microsomes. At the single cardiomyocyte level, VLCAD-/- cardiomyocytes showed significant increase in diastolic Indo-1 and Fura-2 fluorescence, with increased Ca2+ transient amplitude. These changes were associated with altered Ca2+ dynamics, to include: faster sarcomere contraction, larger time derivative of the upstroke, and shorter time-to-minimum sarcomere length compared with VLCAD+/+ control cells. The L-type Ca2+ current characteristics were not different under voltage clamp conditions in the two VLCAD genotypes. SR Ca2+ load measured as normalized integrated NCX current after rapid caffeine application was increased by 48% in VLCAD -/- cells. We conclude that intracellular Ca2+ handling represents a possible molecular mechanism of arrhythmias in mice and perhaps in VLCAD deficient humans.