The DEAD-box RNA helicase p68 plays a very important role in early organ development and maturation. However, the role of p68 in glioma is unclear. In this study, we showed that p68 protein levels were significantly elevated in high-grade gliomas compared to low-grade gliomas and normal adjacent brain tissues. Importantly, the expression of p68 was significantly associated with poorer overall survival and enhanced resistance to treatment with radiotherapy plus temozolomide for glioma patients. Ectopic expression of p68 enhanced glioma cell proliferation both in vitro and in vivo. In contrast, knockdown of endogenous p68 prevented glioma cell proliferation. Using a tandem affinity purification assay, we found a new p68-binding protein, nuclear factor (NF)-κB p50. We found that p68 bound with the N-terminal of NF-κB p50, and the mutant of p68 lacking the p50-interaction domain failed to stimulate glioma cell proliferation and tumor growth. Moreover, p68 induced NF-κB p50 accumulation in the nucleus through release of NF-κB p50 from IκBα and increased NF-κB p50 target luciferase transcription activity. Knockdown of NF-κB p50 rescued the phenotypes induced by p68 both in vitro and in vivo. We concluded that p68 induces glioma tumor growth through binding with NF-κB p50, regulating NF-κB p50 nucleus accumulation and transcription activity.