WW domain-binding protein 2 (WBP2) has been demonstrated as oncogenic in breast cancer. Many studies have revealed the WBP2 gene as a high-risk gene for leukoariaosis and cerebral white matter lesions is important in the pathologic stage of glioma development. This study aimed to illustrate the underlying mechanism by which WBP2 regulates the process of glioma development. The expression pattern of WBP2 in several tumor cells was determined, clarifying the carcinogenic action of WBP2 in glioma cells. Overexpression of WBP2 in glioma cells promoted cell proliferation and migration, and the number of S-phase cells, whereas the depletion of WBP2 by RNAi-mediated knockdown restrained cell growth and cell cycle progression. Upregulation of WBP2 significantly enhanced the tumorigenic ability of U251 cells in vivo. MS/GST pulldown assay identified α-enolase (ENO1) and Homer protein homolog 3 (Homer3) as novel potent interaction partners of WBP2. Knockdown of ENO1 or Homer3 allowed cell growth and migration to return to normal levels. Furthermore, in vitro and in vivo experiments indicated that the oncogenic role of WBP2 in glioma was through modulating ENO1 and glycolysis activity via the ENO1-PI3K/Akt signaling pathway. Collectively, these results reveal that WBP2 plays a vital role in the occurrence and development of glioma, indicating a target gene for glioblastoma treatment.