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Epidermal growth factor receptor (EGFR) had been reported as one of the major responsible genes for malignant progression and phenotype reversion of gliomas, and has been used as one of the most important therapeutic targets. In the present study, small interference RNA (siRNA) and antisense EGFR expression constructs, which target sequences of human EGFR catalytic domain (2400-2420) and the 3′-coding region, respectively, were used to examine the growth inhibition effects on U251 glioma cells. Cell growth was significantly inhibited and G2/M arrest was observed in antisense- and siRNA-treated groups. Matrigel matrix demonstrated spotted cell clustering pattern in antisense- and siRNA-transfected U251 cells, indicating poor cell growth activities. In addition, the tumor volumes in U251 subcutaneous mice model treated with antisense and siRNA were significantly smaller than those treated with control siRNA and phosphate-buffered saline. Also, glial fibrillary acidic protein expression was upregulated in antisense- and siRNA-treated groups than the control groups. Our results demonstrated that antisense- or siRNA-targeting intracellular region of EGFR can inhibit EGFR expression, exerted growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, siRNA expression plasmid-mediated gene therapy would be a new strategy in treatment of gliomas.