Although hepatitis C virus (HCV) causes worldwide health problems, efficient and specific therapy is not available so far. In this study, we describe a new genetic approach to the specific HCV therapy that is based upon trans-splicing ribozymes that can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. We have developed a group I intron-based ribozyme targeting the internal ribosome entry site (IRES) of HCV with high fidelity and specificity. The ribozyme was designed to trans-splice its 3′ tagging sequence comprising a new coding RNA, such as firefly luciferase transcript, that is linked to the 3′ part of the HCV 5′ UTR encompassing the downstream sequence of the targeted residue in the IRES. This ribozyme was then demonstrated to induce HCV IRES-dependent translation of the firefly luciferase gene selectively in HCV IRES-expressing cells with trans-splicing reaction. Moreover, a specific ribozyme with the coding sequence of the diphtheria toxin A chain in place of the firefly luciferase selectively triggered expression of the cytotoxin in cells expressing HCV IRES and specifically activated apoptosis of the cells. These results suggest that the trans-splicing ribozyme could be a potent anti-HCV agent to deliver therapeutic new gene activities specifically and selectively in HCV-infected cells.