Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (> 8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mechanism for mitochondrial dysfunction in lipotoxic cardiomyopathy.