In this study, we found that irradiation in the presence of small interfering RNA-epidermal growth factor receptor (EGFR) arrested U373 glioma cells in G₀ and G₁ phases, delayed cell cycle progression, and effectively inhibited cell proliferation compared with cells that received only radiotherapy. In addition, combined therapy enhanced the percent of apoptotic U373 cells in vitro and also reduced the tumor size and increased the survival rate in tumor xenograft studies. This study demonstrates the antitumor activity of ionizing radiation therapy in combination with small interfering RNA-EGFR in gliomas both in vitro and in vivo and provides a scientific rationale for targeting EGFR to enhance the sensitivity to radiotherapy in patients with glioblastoma multiforme.