Restoration of telomerase activity is essential for most of the malignancies. Telomerase reverse transcriptase (TERT) is the key component of telomerase. In this study, we designed a hammerhead ribozyme against human telomerase reverse transcriptase (hTERT) and observed its growth inhibition and pro-apoptosis effects on cancer cells. The efficiency of this ribozyme was verified in in vitro cleavage experiment. A recombinant retrovirus was constructed to transduce the ribozyme to telomerase positive colon carcinoma cell line SW480 and gastric carcinoma cell line SGC7901. We found that the ribozyme could strongly inhibit hTERT expression and telomerase activity, resulting in rapid apoptosis of cancer cells. Shortening of telomere and replicative senescence were not observed before cell death, indicating intensive inhibition of hTERT expression can induce apoptosis by some mechanism(s) except telomere shortening and replicative senescence. This study suggests that hTERT exerts a direct antiapoptotic function in cancer cells. Anti-hTERT ribozyme might be a potential means in the therapy of telomerase-positive malignancies.