© 2019 American Association for Cancer Research. Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. Significance: LDHA is a pharmacologically tractable EWSFLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.