RLIP76, a GTPase-activating protein, is a central regulator in multiple pathways that respond to redox states and control cell growth, motility, division, and apoptosis in many malignant cancer cells. In this study, human glioblastoma cell lines U87 and U251 were stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting RLIP76. shRNA knockdown of RLIP76 induced cell cycle arrest in U87 and U251 cells and inhibited their invasiveness. Quantitative Western blot analysis revealed that cells stably underexpressing RLIP76 showed lower expression of cyclin D1 and decreased expression and activity of matrix metalloproteinase 2 compared to cells stably transfected with a control vector. Furthermore, RLIP76 expression levels were correlated with IC(50) values for the antitumor drug temozolomide (TMZ). Compared with TMZ alone (17.19 ± 1.78 and 22.18 ± 1.99 μg/mL in U87 and U251 cells, respectively) or combined shGFP and TMZ (18.04 ± 1.07 and 23.040 ± 1.77 μg/mL in U87 and U251 cells, respectively), combined shRNA and TMZ therapy resulted in a significant decrease in IC(50) value (7.61 ± 2.99 and 6.91 ± 2.59 μg/mL in U87 and U251 cells, respectively). Combined RLIP76 knockdown and TMZ treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Furthermore, RLIP76 downregulation enhanced chemosensitivity to TMZ without affecting protein expression of MDR1 and MRP1. The results indicate that inhibition of RLIP76 expression may be an effective means for overcoming RLIP76-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach for glioma treatment.