One of the major concerns with regard to successful cancer gene therapy is to enhance both efficacy and safety. Gene targeting may represent an attractive tool to combat cancer cells without damage to normal cells. Here, we introduce a tumor-targeting approach with the Tetrahymena group I intron-based trans-splicing ribozyme, which cleaves target RNA and trans-ligate an exon tagged at the end of the ribozyme onto the downstream U nucleotide of the cleaved target RNA. We develop a specific trans-splicing ribozyme that can target and reprogram human cytoskeleton-associate protein 2 (hCKAP2)-encoding RNA to trigger therapeutic transgene herpes simplex virus thymidine kinase (HSVtk) selectively in cancer cells that express the RNA. Adenoviral vectors encoding the hCKAP2-specific trans-splicing ribozyme are constructed for in vivo delivery into either subcutaneous tumor xenograft or orthotopically multifocal hepatocarcinoma. We present analyses of the efficacy of the recombinant adenoviral vectors in terms of cancer retardation, target RNA and cell specificity, and in vivo toxicity.