The Proliferating Cell Nuclear Antigen (PCNA), which is an auxiliary protein for DNA polymerase delta, is found to be essential for cellular DNA replication. A designed hammerhead ribozyme, with high efficiency to cleave the PCNA mRNA site-specificly in vitro, was constructed into a self-trimming expression plasmid, and then was introduced into HeLa cells by lipofectin reagent. Small molecular RNAs, isolated from total cellular RNA with the same length as active ribozyme, can cleave the target RNA in vitro, which suggested that this expression plasmid can yield active ribozyme molecules in cells. In comparison with the vector control, the entrance of S phase of the HeLa cells transfected by the ribozyme expression plasmid was delayed 8 hours after serum stimulation. Mean-while, those cells transfected by mutant inactive ribozyme as antisense RNA control was delayed only 3 hours. These results demonstrated that this ribozyme can inhibit the DNA replication in HeLa cells effectively and could be used as a potential tool to study the function of PCNA in cellular DNA replication and cell cycle progression.