Abstract: Background and Objective: Glioma is the most commonly detected primary brain tumor found in adults and has very high rate of morbidity and mortality. The present study was performed to investigate the effect of gedunin on viability, migration and invasion of human glioblastoma multiforme, U-251 MG cell line. Materials and Methods: Invasion assay was performed using 24-well cell culture invasion chamber (Corning Inc., Tewksbury, MA, USA) coated with 8.0 μm Matrigel™ (Becton Dickinson, Bedford, MA, USA). For real-time polymerase chain reaction U-251 MG cells were distributed on to the six-well tissue culture plates and treated with gedunin for 24 h. Qiagen RNeasy Mini Kit was used for the isolation of total RNA from the cells. Results: The results revealed that exposure of U-251 MG cells to gedunin reduced the viability of U-251 MG cells to 28% compared to 98% in control group. Analysis of cell migration and invasion showed a significant inhibition by gedunin at a concentration of 20 μM after 24 h. Gedunin treatment suppressed the expression of focal adhesion kinase (FAK), metalloproteinases (MMP)-7, MMP-9 and uPA which play a vital role in inducing migration of carcinoma cells. The inhibition was significant (p<0.005) at all the tested concentrations except 5 μM after 24 h in U-251 MG cells. In addition the real-time PCR analysis revealed a marked reduction in the expression of MMP-9, FAK and Rho kinases-ROCK-1 (ROCK-1) mRNA by gedunin treatment. Conclusion: Thus, gedunin treatment caused inhibition of proliferation, migration and invasion in U-251 MG cells through inhibition of MMP-9, FAK and ROCK-1. Therefore, gedunin is a potent agent for the treatment of tumor growth.