Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (ip) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.