Survivin is a new member of the inhibitor of apoptosis protein (IAP) family that is implicated in the control of cell proliferation and the regulation of cell life span. This protein is selectively expressed in most human carcinomas but not in normal adult tissues. To down-regulate a human survivin expression as a strategy for cancer gene therapy, we designed two hammerhead ribozymes (RZ-1, RZ-2) targeting human survivin mRNA. RZ-1 and RZ-2 efficiently cleaved the human survivin mRNA at nucleotide positions +279 and +289, which was identified by in vitro cleavage assay using in vitro transcribed ribozymes and truncated survivin mRNA substrate. To investigate the function of the ribozymes in cells, the sequences of the ribozymes were cloned into replication-deficient adenoviral vector and transferred to breast cancer cell, MCF-7. The infection with adenovirus encoding the ribozymes resulted in a significant reduction of survivin mRNA (74% and 73%, respectively) and protein. As revealed by nuclear condensation/ fragmentation and flow cytometry analysis, inhibition of survivin gene by ribozymes increased apoptosis and sensitivity induced by etoposide or serum starvation. Our results suggest that the designed hammerhead ribozymes against survivin mRNA are good candidates for feasible gene therapy in the treatment of cancer.