The ERK and PI3K signaling cascades are aberrantly activated in human glioblastoma cells, resulting in the dysregulation of numerous downstream transcription factors. The dark side of the transcription factor, NF-E2-related factor 2 (Nrf2) in human cancer has been revealed. It has been accepted that high levels of Nrf2 promote tumor progression. In the present study, we investigated the effect of the ERK and PI3K signaling cascades on Nrf2 in human glioblastoma cells. Immunohistochemical staining for Nrf2 in clinical specimens showed that the expression and nuclear localization of Nrf2 were increased in human glioblastoma tissues when compared to peritumoral normal tissues. In addition, we detected decreased nuclear localization of Nrf2 following combined treatment with ERK and PI3K inhibitors in three human glioblastoma cell lines and selected the cell line (U251) most sensitive to the inhibitors for further study. Our data demonstrated that inhibition of ERK and PI3K not only suppressed the nuclear accumulation of Nrf2 protein but also decreased the expression of the Nrf2 protein. In addition, combined inhibition of ERK and PI3K also decreased the mRNA levels of Nrf2 target genes. Finally, we found that Nrf2 overexpression partly reversed the ERK and PI3K inhibitor-induced inhibition of cell viability. Therefore, the ERK and PI3K signaling cascades regulate the expression and activation of Nrf2 and control cell viability partly through Nrf2 in U251 human glioblastoma cells. Thus, targeting the ERK and PI3K signaling cascades for Nrf2 activation may provide new methods for the treatment of glioblastoma.