Targeting cell metabolism has emerged as a viable treatment strategy in acute myeloid leukemia (AML), a malignant hematological disease characterized by poor patient outcomes and limited chemotherapeutic options. Compared to the normal hematopoietic population, leukemia cells exhibit an altered phenotype characterized by increased mitochondrial mass as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. Mitochondrial FAO is a four-reaction process that catabolizes fatty acids to acetyl-CoA, generating reductive equivalents for the electron transport chain (ETC) and anaplerotic intermediates for the TCA cycle. Clinically approved FAO inhibitors, such as trimetazidine and ranolazine, are tissue-specific, targeting FAO in some tissues but affecting additional pathways in others. To better understand the potential clinical utility of targeting FAO, we systematically tested a panel of clinical and pre-clinical FAO inhibitors, reasoning that the most potent FAO inhibitor would lead to a novel anti-AML target. Avocadyne was the most potent anti-AML compound, inducing leukemic cell death (EC50: 2.5 µM) and suppressing clonogenic growth of primary samples, while sparing normal hematopoietic cells. Further, avocadyne (100mg/kg twice weekly for 5 weeks) reduced patient-derived AML cell engraftment in the bone marrow of immune deficient mice. As a component of avocatin-B, a mixture of two fatty alcohols previously determined to accumulate in the mitochondria, we confirmed that avocadyne inhibited long chain FAO using radiolabeled studies and high resolution respirometry. To identify a molecular target, avocadyne treated cells were immunoprecipitated with antibodies against each intramitochondrial enzyme involved in long chain FAO (e.g., very long acyl-CoA dehydrogenase (VLCAD; step 1) and the alpha and beta subunits of the mitochondrial trifunctional protein: HADHA, HADHB; steps 2-4). Immunoblotting and LC/MS analysis confirmed that avocadyne co-eluted with VLCAD, but not with HADHA or HADHB, confirming a direct physical interaction between avocadyne and VLCAD. VLCAD introduces a double bond to a fully saturated long chain fatty acid and reduces electron transfer flavoprotein (ETF)-bound FAD, transferring these electrons to the ETC. Using fluorescence spectrophotometry and respirometry, avocadyne directly inhibited VLCAD activity, resulting in reduced ETF-supported respiration. The activity of MCAD, an acyl-CoA dehydrogenase catalyzing medium chain fats, was not affected, suggesting avocadyne inhibits long chain FAO exclusively. Further, profiling of acyl-carnitines following avocadyne treatment also showed a pattern characteristic of long chain FAO inhibition at VLCAD. To further understand how VLCAD modulated avocadyne sensitivity, avocadyne-resistant and VLCAD knockdown cells were generated. Lentiviral knockdown of VLCAD sensitized leukemic cells to avocadyne-induced FAO inhibition and death. In contrast, cells resistant to avocadyne had increased protein expression of VLCAD but no change in other long chain FAO enzymes. With increased VLCAD-supported ETF respiration, higher concentrations of avocadyne were required to induce cell death, compared to the parental line. These results show that VLCAD expression modulated leukemic sensitivity to avocadyne. Inhibiting FAO at VLCAD triggered an adaptive metabolic switch towards glycolysis, characterized by increased extracellular acidification. This compensatory increase in glycolysis was ultimately insufficient to prevent the depletion of TCA metabolites and ATP, leading to leukemic death. In contrast, following avocadyne treatment, normal umbilical cord blood-derived mononuclear cells increased glycolytic as well as pyruvate dehydrogenase activity and had no decrease in ATP levels, cell viability, or clonogenic growth. Together, these results highlight, for the first time, VLCAD as a novel anti-AML target and further suggest the clinical utility of FAO inhibition as a potential anti-AML strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.