Oxidative stress could induce apoptosis and autophagy process simultaneously, but the role of autophagy is still not clear. Beclin 1, a key gene regulating the preautophagosome formation, is involved in the injury induced by oxidative stress. To observe the role of autophagy in H2O2-induced injury of U251 cells, the recombinant plasmid Psilencer3.1-siRNA-Beclin 1 was transfected into U251 cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control groups respectively. The cells were collected 24 h later, and the cell total protein was extracted to detect Beclin 1, Bcl-2 and Bax protein expressions by Western blot. After the Beclin 1-siRNA cells were treated with 1 mmol/L H2O2, the autophagic vacuoles in the cells were stained with monodansylcadaverine (MDC), and the cell apoptotic ratio was determined with PI/Annexin V-FITC staining by flow cytometry analysis. The results showed that the synthetic siRNA decreased the expression of Beclin 1 protein significantly, but had no obvious effect on the levels of Bcl-2 and Bax protein expressions. Compared with those in the control group, the autophagic vacuoles, the level of LC3-II protein expression and the percentage of apoptotic cells increased (P < 0.05) in 1 mmol/L H2O2 group. In Beclin 1-siRNA + H2O2 group, autophagic vacuoles and the levels of LC3-II protein expression decreased obviously, the percentage of apoptotic cells increased significantly compared with that in 1 mmol/L H2O2 group (P < 0.05). H2O2 and autophagy inhibitor 3-methyladenine (3-MA) combination also increased the percentage of apoptotic cells obviously (P < 0.05). These results revealed that the transfection of Psilencer3.1-siRNA-Beclin 1 effectively inhibited the expression of Beclin 1 protein expression, degraded the autophagy level and increased the apoptotic rate in U251 cells under oxidative stress, which was coincident with the effect of autophagy inhibitor 3-MA. This study suggests that autophagy is a cell protective role in oxidative stress process, and the inhibition of autophagy may enhance apoptosis.