Notch signaling plays a pivotal role in many cancers, including glioblastoma (GBM). Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) is a key transcription factor of the Notch signaling pathway. Here, we interrogated the function of RBPJ in GBM. Firstly, RBPJ expression of GBM samples was examined. Then, we knocked down RBPJ expression in 2 GBM cell lines (U251 and T98) and 4 glioblastoma (GBM) stem-like cell lines derived from surgical samples of GBM (KGS01, KGS07, KGS10 and KGS15) to investigate the effect on cell proliferation, invasion, stemness, and tumor formation ability. Expression of possible downstream targets of RBPJ was also assessed. RBPJ was overexpressed in the GBM samples, downregulation of RBPJ reduced cell proliferation and the invasion ability of U251 and T98 cells and cell proliferation ability and stemness of glioblastoma stem-like cells (GSC) lines. These were accompanied by reduced IL-6 expression, reduced activation of STAT3, and inhibited proneural-mesenchymal transition (PMT). Tumor formation and PMT were also impaired by RBPJ knockdown in vivo. In conclusion, RBPJ promotes cell proliferation, invasion, stemness, and tumor initiation ability in GBM cells through enhanced activation of IL-6-STAT3 pathway and PMT, inhibition of RBPJ may constitute a prospective treatment for GBM.