Telomerase is highly expressed in advanced stages of most cancers where it allows the clonal expansion of transformed cells by counteracting telomere erosion. Telomerase may also contribute to tumor progression through still undefined cell growth-promoting functions. Here, we inhibited telomerase activity in 2 human glioblastoma (GBM) cell lines, TB10 and U87MG, by targeting the catalytic subunit, hTERT, via stable RNA interference (RNAi). Although the reduction in telomerase activity had no effect on GBM cell growth in vitro, the development of tumors in subcutaneously and intracranially grafted nude mice was significantly inhibited by antitelomerase RNAi. The in vivo effect was observed within a relatively small number of population doublings, suggesting that telomerase inhibition may hinder cancer cell growth in vivo prior to a substantial shortening of telomere length. Tumor xenografts that arose from telomerase-inhibited GBM cells also showed a less-malignant phenotype due both to the absence of massive necrosis and to reduced angiogenesis. © 2005 Wiley-Liss, Inc.