Although hepatocellular carcinoma (HCC) is one of the world-wide common malignancies, development of more specific and controlled therapeutic methods should be warranted. In this study, we describe a novel approach to HCC therapy that is based on trans-splicing ribozyme-mediated replacement of HCC-associated specific RNAs. We have developed a specific ribozyme that can target and replace human alpha-fetoprotein (AFP) RNA, which is highly expressed in HCC, with new transcript exerting therapeutic activity selectively in AFP-expressing liver cancer cells. The RNA replacement was employed via a high-fidelity trans-splicing reaction with the targeted residue in the AFP-expressing cells. Noticeably, the ribozyme could selectively deliver activity of suicide gene, herpes simplex virus thymidine kinase gene, into the liver cancer cells expressing the AFP RNA and thereby specifically and effectively retarded the survival of these cells with ganciclovir treatment. Simultaneously with the specific induction of therapeutic gene activity, the ribozyme reduced expression level of the targeted AFP RNA in the cells. These results suggest that the AFP RNA-targeting trans-splicing ribozyme could be a useful genetic agent for HCC-targeted efficient gene therapy.