Membrane contact sites between organelles are critical for transfer of biomolecules. Lipid droplets (LDs) store fatty acids and form contacts with mitochondria, which regulate fatty acid oxidation and energy ATP production. The interaction between LDs and mitochondria are increased during times of heightened energy demand, such as adrenergic stimulation or exercise, and during states of increased reliance on fatty acid oxidation, such as starvation and cold adaptation. Protein compartmentalization at LD-mitochondria contact sites and their effects on biological processes are poorly described. Using split-BioID proximity labeling, we identified 71 proteins at LD-mitochondria contact sites, many of which are known ER localized proteins. These include a multimeric complex containing extended synaptotagmin (ESYT) 1, ESYT2, and VAMP Associated Protein B and C (VAPB) that transfers fatty acids to enable beta-oxidation. Depletion of ESYT1, ESYT2 or VAPB limits LD-derived fatty acid oxidation, resulting in depletion of TCA cycle metabolites, remodelling of the cellular lipidome, and induction of lipotoxic stress. These findings were recapitulated with Esyt1 or Esyt2 deletion in livers of mice. This study identified proteins residing at LD-mitochondria-ER contact sites and uncovered a fundamental mechanism for fatty acid transfer required for cellular lipid homeostasis, with implications for metabolic diseases and survival.