Abstract Body (Do not enter title and authors here): Mammalian cells have several adaptive mechanisms to counteract the adverse effects of metabolic and redox stress induced by ischemia. Hypoxia, a hallmark of ischemia, results in the selective reduction of the tricarboxylic acid cycle metabolite, α-ketoglutarate, to the L-(S)-enantiomer of 2-hydroxyglutarate (L2HG) in several cell types. L2HG protects hypoxic cells by buffering increases in the NADH/NAD⁺ redox couple and inhibiting mitochondrial electron transport. In addition, the accumulation of L2HG induced by genetic knockout of L2HG dehydrogenase (L2HGDH), the only known enzyme capable of oxidizing L2HG back to α-ketoglutarate, protects mice against myocardial injury during ischemia. This protection by L2HG manifests as decreased myocardial infarct size and improved cardiac function, owing partly to a metabolic shift in carbon flux from glycolysis towards the pentose phosphate pathway. However, myocardial ischemia also leads to perturbations in fatty acid metabolism as manifest by accumulation of acylcarnitines and acyl-CoA’s. It remains unclear as to whether or not L2HG accumulation affects fatty acid metabolic homeostasis during myocardial ischemia. Here, we induced L2HG accumulation by homozygous deletion of the l2hgdh gene in male mice (l2hgdh^-/-; n=12). Hearts isolated from these mice and their wild-type littermates (l2hgdh^+/+; n=13) were subjected to ex vivo Langendorff perfusion at coronary perfusion pressure of 80 mmHg for 30 min with (ischemic group) or without (control group) subsequent perfusion at ~10% pressure for 90 min. Using liquid chromatography tandem mass spectrometry (LC-MS/MS)-based nontargeted lipidomics, we identified several species of long chain acylcarnitines that accumulated by ischemia in hearts obtained from L2HGDH^+/+ mice [linoleoyl carnitine, palmitoyl carnitine, and hydroxy-linoleoyl carnitine increased by 13.2-fold (p=4.2 x 10^-6), 10.7-fold (p=1.7 x 10^-6), and 10.0-fold (p=5.8 x 10^-6), respectively]. Interestingly, however, this accumulation was attenuated substantially in ischemic hearts obtained from L2HGDH^-/- mice [hydroxy-linoleoyl carnitine, palmitoyl carnitine, and linoleoyl carnitine were downregulated by 62.6% (p=0.034), 54.2% (p=0.018), and 35.4% (p=0.054) in ischemic hearts from L2HGDH^-/- mice when compared to L2HGD^+/+ littermates]. Overall, these findings highlight a novel and potentially important role for L2HG in restoring the perturbations in fatty acid metabolism induced by myocardial ischemia.