To construct erythroblastic leukemia viral oncogene homolog 2 (ErbB2) small interference RNA ( siRNA) expression vector and to study its effect on U251 cell line proliferation and apoptosis combining with ,60,Co γ-iradiation. ErbB2 specific 19bp oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form the double strand DNA fragments,which was cloned into pSilence2.1-U6-H1 vector. The recombinant pSilence2.1-ErbB2 expression construct was confirmed by Hind III and BamH I double digestion and sequencing. The pSilence 2.1-ErbB2 was transfected into U251 cell. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry. The apoptosis of transfected U251 cell was examined with Hoechst 33258 staining and Annexin-V kit. Psilence 2.1-ErbB2 expression vector was successfully constructed and it can effectively inhibit proliferation(,p,<,0.05) and induced time dependent apoptosis(the percentage of apoptosis cell increase 10.52% after 24h transfection, the percentage of apoptotic cell increase 50.65% after 48h transfection) in transfected U251 cell line compared with non-transfected and pSilence2.1-GFP U251 cells, After combination with ,60,Co γ-irradiation, the effect of inhibiting proliferation was more significant compared with non-irradiated U251 cells(,p,<,0.05). The pSilence 2.1-ErbB2 expression vector can effectively inhibit proliferation and induced U251 cells time dependent apoptosis; combination of ,60,Co γ-irradiation can enhance the inhibitory efficiency in U251 cell line.