Protein-mediated lipid trafficking between organellar membranes is a key regulator of cellular metabolism, bioenergetics, calcium signaling, immunity, and cell death. Lipid droplets (LDs) are the reservoir for fatty acid storage and mitochondria mediate fatty acid oxidation and ATP production through β-oxidation and oxidative phosphorylation. The process by which fatty acids is transferred between these organelles has remained elusive. Here, we used split BioID proximity labelling to characterize the proteins that reside at LD-mitochondria contact sites. Of the 74 identified proteins, we demonstrate that extended synaptotagmin (ESYT) 1, ESYT2, and VAPB form multimeric complexes required for trafficking of fatty acids from LDs to the mitochondria for β-oxidation. Lipid docking and mechanistic analyses indicate that the lipid-binding pocket in ESYT1 and ESYT2 is capable of transferring medium- and long-chain fatty acids. Depletion of ESYT1, ESYT2 or VAPB limits LD-derived fatty acid oxidation, resulting in enhanced lipid storage in enlarged lipid droplets and induction of lipotoxic cellular stress pathways. These findings were capitulated in mice with hepatocyte-specific silencing of Esyt1 or Esyt2 and fat-body specific deletion of Esyt2 in Drosophila reduced survival in response to starvation. Together, these findings suggest that ESYT1/ESYT2/VAPB mediated fatty acid transfer at LD-mitochondria contact sites is essential for the maintenance of cellular lipid homeostasis.