Hypoxia is intimately associated with enhanced glycolysis in gliomas, and hypoxia-inducible factor 1α (HIF-1α) plays a critical role in this process. RLIP76 (Ral-interacting protein 76) functions as a multifunctional mediator and is aberrantly expressed in various malignant tumors, including glioma. However, the underlying mechanism of RLIP76 and HIF-1α in glioma glycolysis remains largely unclear. In the present study, we demonstrated that RLIP76 is a hypoxia-inducible molecule that contributes to facilitating glycolysis in glioma cells under hypoxic conditions. In addition, hypoxia-induced RLIP76 is a novel target of HIF-1α and enhances the two important HIF-1α-target glycolytic proteins glucose transporter type 1 (GLUT1) and lactate dehydrogenase A (LDHA) in hypoxia. Mechanistically, RLIP76 can directly bind to HIF-1α in the nucleus and regulate the stability of HIF-1α by alleviating HIF-1α ubiquitination and therefore activates GLUT1 and LDHA to accelerate glycolysis in hypoxia. Furthermore, the enhanced glycolysis is necessary for the role of RLIP76 to promote glioma development in vivo, confirming the ability of RLIP76 to regulate tumor cell glycolysis. Collectively, our results demonstrate a previously unappreciated function of RLIP76 in hypoxia-mediated glycolytic metabolism and implicate that RLIP76 might be a valuable therapeutic target for gliomas.