Telomerase is implicated in the development of cellular immortality and oncogenesis. It has been shown that telomerase activity is considerably higher in the tissue of many different cancers than in normal tissue, and that the inhibition or downregulation of telomerase activity can prevent the malignant proliferation of tumor cells. Antisense oligonucleotides have been widely used in suppressing the expression of genes and, therefore, in the present research, we evaluated the effect of antisense human telomerase RNA (hTR) on glioma cell growth in vitro and in vivo. We showed that antisense hTR cDNA significantly inhibited TJ905 human glioma cell proliferation in vitro and tumor growth in vivo, as determined by MTT assay and by measuring the volume of glioma in nude mice. Consistent with these results, we found that telomerase activity and the mRNA levels of hTR and hTERT (human telomerase reverse transcriptase) expression were markedly decreased in tumor cells treated with antisense hTR cDNA, as assessed by TRAP (telomeric repeat amplification protocol) assay and RT-PCR (reverse transcription-polymerase chain reaction) analysis. Our study conclusively demonstrates that antisense hTR effectively inhibits the growth of human glioma cells in vitro and in vivo and, thus, may be potentially used for gene therapy of malignant gliomas and other cancers.