Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.