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Results We found that DHA prevented cell proliferation in K562 cells through inhibiting aerobic glycolysis. Lactate product and glucose uptake were inhibited after DHA treatment. Results showed that DHA modulates glucose uptake through downregulating glucose transporter 1 (GLUT1) in both gene and protein levels. The cytotoxicity of DHA on K562 cells was significantly reversed by PKM2 agonist DASA-58. Pyruvate kinase activity was significantly reduced after DHA treatment, decreased expression of PKM2 was confirmed in situ. Conclusion The