Most cancer cells exhibit a shift in glucose metabolic strategy, displaying increased glycolysis even with adequate oxygen supply. SUMO-specific proteases (SENPs) de-SUMOylate substrates including HIF1α and p53,two key regulators in cancer glucose metabolism, to regulate their activity, stability and subcellular localization. However, the role of SENPs in tumor glucose metabolism remains unclear. Here we report that SUMO-specific protease 2 (SENP2) negatively regulates aerobic glycolysis in MCF7 and MEF cells. Over-expression of SENP2 reduces the glucose uptake and lactate production, increasing the cellular ATP levels in MCF7 cells, while SENP2 knockout MEF cells show increased glucose uptake and lactate production along with the decreased ATP levels. Consistently, the MCF7 cells over-expressing SENP2 exhibit decreased expression levels of key glycolytic enzymes and an increased rate of glucose oxidation compared with control MCF7 cells, indicating inhibited glycolysis but enhanced oxidative mitochondrial respiration. Moreover, SENP2 over-expressing MCF7 cells demonstrated a reduced amount of phosphorylated AKT, whereas SENP2 knockout MEFs exhibit increased levels of phosphorylated AKT. Furthermore, inhibiting AKT phosphorylation by LY294002 rescued the phenotype induced by SENP2 deficiency in MEFs. In conclusion, SENP2 represses glycolysis and shifts glucose metabolic strategy, in part through inhibition of AKT phosphorylation. Our study reveals a novel function of SENP2 in regulating glucose metabolism.