The over-expression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/deletion of tumor suppressor PTEN gene are main genetic changes identified in glioblastomas. These two genetic changes play a critical role in the formation of many malignant tumors and have been shown to be the important therapeutic targets. In this study, we used an expression plasmid that expresses small hairpin RNA-targeting sequences of human EGFR and wild-type PTEN cDNA to examine the growth inhibitive effects in U251 glioma cells. It was found that down-regulation of EGFR expression and up-regulation of PTEN expression resulted in the suppression of cell proliferation, arrest of cell cycle, reduction in cell invasion and promotion of cell apoptosis in vitro. In addition, the growth of the subcutaneous U251 glioma in the nude mice treated with expression plasmid was significantly inhibited. Our results demonstrated that the expression plasmid could exert proliferation and invasion inhibition effects on U251 cells in vitro and in vivo. It suggested that combinatory gene therapy targeting EGFR and PTEN would be a new strategy in gene therapy of glioblastoma.