Fat is a vital source of fuel for many organs, and the processes governing its metabolism have been studied in numerous contexts over the past century. However, still little is known as to how the circulating lipids traverse the capillary wall in order to be taken up by the underlying parenchyma. This is especially relevant in tissues whose capillary networks consist of tightly bound and continuous endothelial cells (ECs). There is great clinical value in parsing out these mechanisms, as excessive lipid accumulation in the skeletal muscle leads to insulin resistance and type 2 diabetes (T2DM). Recent work from our lab has identified that 3-hydroxyisobutyrate (3-HIB), which has been previously implicated as a biomarker for T2DM, is secreted by the muscle and induces endothelial fatty acid (FA) transport. Interestingly, I have since found that 3-HIB is not unique in its effect; certain other similarly structured compounds such as lactate can also stimulate FA uptake. However, the mechanisms controlling even basal FA uptake remain understudied. To gain a better understanding of endothelial lipid handing, we conducted a chemical screen that demonstrated the mitochondrial uncoupler niclosamide as a potent inhibitor of endothelial FA uptake and transport. Further work expanding on this discovery revealed that mitochondrial ATP production regulates endothelial FA uptake. This was unexpected, as most ATP generated in ECs are derived from glycolysis, suppression of which has no effect on FA uptake. Using confocal microscopy and immunostaining, we have determined that the mitochondrial ATP is likely being used in mitochondrial/ER microdomains, where fatty acid transport 4 (FATP4) employs its ATP-dependent acyl-CoA synthetase function to trap and activate incoming FAs, driving further FA uptake via vectorial acylation. Furthermore, endothelial mitochondria may be playing a role in 3-HIB or lactate stimulated FA uptake as well. These findings have given us surprising insight into the regulation of endothelial FA uptake and provide new potential avenues for therapeutic intervention against metabolic disease.