Abstract: ObjectiveTo explore the effects of Dis3l2 knockout on the glucose metabolism of gastric cancer cells. MethodsCRISPR/Cas 9 system was used to knock out Dis3l2 gene. The amount of lactic acid in the culture media was measured by biological methods. The level of phosphate fructose kinase (PFK) in the cells was also detected by ELISA. The levels of mammalian target of rapamycin (mTOR), hypoxia inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT-1), and pyruvate kinase M2 (PKM2) were detected using Western blotting. ResultsKnockout of Dis3l2 in gastric cancer cells resulted in increases in the level of lactic acid in the culture media and PFK levels in the cells, while up-regulating the expression of mTOR, HIF-1α, GLUT-1, and PKM2. ConclusionKnockout of Dis3l2 promotes the glycolysis of gastric cancer cells, which may be related with the up-regulation of HIF-1α, GLUT-1, and PKM2 in the mTOR pathway.