In order to explore the function of PCNA gene on the proliferation and apoptosis of hepatocelluar carcinoma HepG2 cells, to provide the experimental evidence and a new tool to further explore the function of PCNA gene and the feasibility of its gene therapy, the eukaryotic expression vector targeting PCNA was constructed. According to the sequence screened in previous work, PCNA siRNA was converted into cDNA coding expression of shRNA (small hairpin RNA). The cDNA was synthesized and inserted into plasmid pSilencer2.0-U6 to construction of eukaryotic expression vector of siRNA specific for targeting PCNA gene, the negative plasmid pShPNA was also constructed. The plasmids were identified by sequence analysis. The plasmid was transiently transfected into hepatocelluar carcinoma HepG2 cells for 48 h, proliferation and the clone formation of HepG2 cells were detected by CCK-8 and clone formation assay respectively. The percentage of hypodiploid cells and early apoptotic cells was detected by flow cytometry. The morphology was examined by fluorescence microscope after Hoechst 33258 staining. Compared with control group and negative control group, PCNA mRNA level was reduced by pShPCNA detected by RT-PCR. The proliferation and clone formation of HepG2 cells treated with pShPCNA for 48 h were significantly inhibited (P < 0.01). The migration was also attenuated when PCNA was knocked down by shRNA, and flow cytometry analysis showed an increase of the percentage of G0/G1 phase cells, along with a decrease of cell population in the S phase. Percentages of hypodiploid cells and early apoptosis rates were significantly higher in treatment groups than those in control and negative control group (P < 0.01). Mitochondrial membrane potential was reduced in pShPCNA group detected by JC-1 fluorescent staining. Apoptotic morphology such as cell shrinkage, nuclear condensation, nuclear fragmentation, chromatin condensation and apoptotic bodies were also observed by staining with Hoechst33258 under fluorescence microscope. The study indicated the eukaryotic expression vector, PCNA shRNA has been successfully constructed, and effectively inhibits proliferation and induces apoptosis and arrested HepG2 cells in G0/G1 phage.