Glioblastoma multiforme (GBM), the most common type of central nervous system tumor in humans, is highly proliferative and resistant to apoptosis associated with genetic mutations that deregulate cell cycle. Signal transduction and activation of transcription 3 (Stat3) is a key signal transduction protein that mediated signaling by cytokines and contributed to oncogenesis. It is constitutively activated in numerous cancers including glioblastoma. To determine the effect on proliferation and differentiation of glioblastoma U251 cells after inhibiting STAT3 expression by RNAi, STAT3 gene was silenced with lentivirus vector in U251 cells. We demonstrate that a lentivirus-based shRNA vector had highly infecting efficiency to U251 cells and lentivirus vector-mediated RNAi significantly suppressed Stat3 expression and activation in U251 cells. Knockdown of STAT3 expression by RNAi suppressed the growth and induced apoptosis of U251 cells by down-regulating expression of Bcl-2. It was found that the cell proportion of G0/G1 phase significantly increased after silencing Stat3 by down-regulating expression of cyclin D1. Knockdown of Stat3 also induces morphological changes of U251 cell. It increases significantly expression of myelin basic protein (MBP) in U251 cells. This study demonstrates that STAT3 silencing with lentivirus effectively inhibits STAT3 gene expression and activation. Stat3 is associated with the survival, growth and differentiation of U251 cells. Lentivirus vector-mediated RNAi may be serve as a novel therapeutic strategy for treatment of GBM with expression constitutively and activation of STAT3 gene.