The cysteine proteinase cathepsin B has been implicated in tumor progression by virtue of its increased mRNA and protein levels, as well as its localization at the invading front of the tumor. In this study, we examined whether blocking cathepsin B expression in human glioblastoma SNB19 cells affects angiogenesis. Stable transfectants of human glioblastoma cells with a plasmid containing antisense cathepsin B cDNA showed decreased migration rates in wound- and spheroid-migration assays. Analysis showed a reduction in VEGF protein and MMP-9 activity in the cathepsin B antisense cDNA-transfected cells. Regarding angiogenesis in vitro, we found that the conditioned medium of glioblastoma cells with downregulated cathepsin B expression reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary-like network formation. Furthermore, a marked reduction in microvasculature development was seen in an in vivo dorsal air sac assay of glioblastoma cells with downregulated cathepsin B expression. Taken together, these results provide evidence that inhibition of cathepsin B expression can suppress glioblastoma-induced neovascularization.