Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions. Display omitted •Induction of EndoMT triggers a reduction in FAO•FAO is required to maintain endothelial acetyl-CoA levels•FAO modulates in vitro and in vivo EndoMT Xiong et al. demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of endothelial-to-mesenchymal transition (EndoMT) and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.