Many efforts have been taken to develop molecule target for cancer therapy. 14-3-3zeta protein has emerged as a critical regulator of diverse cellular pathways in multiple cancers. Furthermore, 14-3-3zeta expression was elevated and a predictor of poor prognosis in glioblastoma. However, there is no information to evaluate the potential effects of 14-3-3zeta RNAi in glioblastoma. The relationship between 14-3-3zeta expression and cell proliferation and apoptosis was tested in primary glioblastoma samples. Through an RNAi approach using human glioblastoma cells as a model system, we demonstrated the role of 14-3-3zeta in glioblastoma proliferation, apoptosis, invasion and tumor growth. The expression of 14-3-3zeta in glioblastoma stem cells was also investigated by immunostaining. The apoptosis was significantly higher in 14-3-3zeta-negative group than in positive group. 14-3-3zeta immunoreactivity score was negatively correlated with the apoptosis, and positively with proliferation in human specimens. 14-3-3zeta RNAi reduced cell proliferation, induced apoptosis, decreased the invasive capability and colony-formation, and impaired the growth of glioblastoma xenografts in nude mice. Moreover, 14-3-3zeta was positively expressed in glioblastoma stem cells. Our data highlight the importance of 14-3-3zeta in glioblastoma and identify 14-3-3zeta as a potential molecular target for glioblastoma treatment.