Taurine-upregulated gene 1 (TUG1) has been reported as an oncogenic long non-coding RNA (lncRNA) in acute myeloid leukemia (AML). Nevertheless, the roles and molecular mechanism of TUG1 in drug resistance of AML cells are still unclear. Glycolysis level was evaluated by detecting glucose consumption and lactate production. qRT-PCR and Western blot were performed to detect TUG1, hexokinase2 (HK2) and pyruvate kinase isoenzyme M2 (PKM2) expressions. Adriamycin (ADR) cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The changes of the protein kinase B (Akt) pathway were determined by Western blot analysis of phosphorylated-Akt (p-Akt) (ser473) and Akt. Our results showed that glycolysis was increased in HL60/ADR cells, as evidenced by the elevated glucose consumption and lactate production, as well as the increased HK2 and PKM2 expressions at mRNA and protein levels. TUG1 was up-regulated in HL60/ADR cells and TUG knockdown inhibited glycolysis. TUG1 knockdown enhanced ADR-induced cytotoxicity and apoptosis in HL60/ADR cells. TUG1 knockdown inhibited the Akt pathway and activation of the Akt pathway by 740Y-P attenuated the effects of TUG1 knockdown on ADR-induced cytotoxicity and apoptosis, as well as glycolysis in HL60/ADR cells. Taken together, TUG1 knockdown enhances adriamycin cytotoxicity in HL60/ADR cells via inhibiting the glycolysis by inactivating the Akt pathway.