Abstract: To observe the effect of short hairpin RNA (shRNA) targeting the survivin gene on apoptosis of human brain glioblastoma U251 cells in vitro. Methods: For the U251 cells, U251- SR cells stably transfected with the shRNA eukaryotic expression vector pWH1- SR targeting the survivin gene, and U251- P cells stably transfected with the empty pWH1 vector, phase contrast microscopy,H&E staining, Hoechst staining, TUNEL staining and transmission electron microscopy were used to observe the morphologic characteristics of the cells in each group. Flow cytometry (FCM) was used to determine the number of apoptotic cells in each group. Results: The results of phase contrast microscopy, H&E staining, Hoechst staining, TUNEL staining and transmission electron microscopy showed that the percentage of apoptotic cells among the U251- SR cells significantly increased, producing morphologic changes typical of apoptosis. The FCM quantitative analysis showed that the U251- SR apoptotic cells amounted to 14.4% of the total population, an increase of about 6- fold, compared with the U251 (2.1%) and U251- P (2.7%) cells. Conclusion: shRNA targeting the survivin gene can induce apoptosis of U251 cells in vitro.