Eps8 was initially identified as a substrate of the epidermal growth factor receptor. Overexpression of Eps8 leads to increased mitogenic signaling and malignant transformation. However, little is known concerning the importance of Eps8 in human gliomas. In this study, we found that Eps8 was overexpressed in 56.6% of human gliomas (WHO grades III and IV) compared with adjacent normal brain tissues by immunohistochemical analysis. The U251 human glioma cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced U251 glioma cell growth and survival by cell survival, MTT and liquid colony formation assays. By contrast, the lentiviral expression of Eps8 siRNA in SHG-44 cells resulted in a significant reduction in cellular growth and proliferation. Furthermore, Eps8 modulated the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), phosphorylated serine-threonine protein kinase Akt and β-catenin expression in glioma cell lines and tissues. These results suggest that Eps8 is overexpressed in human gliomas, and affects glioma cell growth possibly by regulating ERK and Akt/β-catenin signaling. Therefore, Eps8 may represent a novel potential target in human glioma therapy.