CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133+-C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133+-C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133+-C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133+-C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133+-C6. A decline in the invasion of hCD133+-C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133+-C6. In vivo study further showed that hCD133+-C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133+-C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133+-C6 in vitro, as well as progressive tumor formation in vivo.