Abstract: Objective To construct short hairpin RNA (shRNA) expression plasmids targeting at the cortactin gene, and to screen the plasmid with the most knockdown efficiency. Methods Three pairs of shRNAs targeting at the cortactin gene were designed and synthesized. The shRNA expression plasmids (named pBSilence1.1-cortactin-shRNA1-3) were constructed and identified using restriction enzyme analysis and sequence analysis. The plasmids (pBSilence1.1-cortactin-shRNA1-3 and negative control plasmid) were then transfected into HepG2 cells via liposome. The transfection results were observed after 24h, and the cortactin mRNA expression was measured by reverse transcriptase-polymerase chain reaction(RT-PCR). Results The expression plasmids were confirmed by restriction enzyme analysis and sequence analysis. After the plasmid vector with green fluorescent protein was transfected to the cells, it was seen as a green fluorescent wave. RTPCR results showed that the cortactin mRNA expression in HepG2 cells of the three recombinant plasmids groups were decreased significantly compared with the negative control and blank control group(P＜0.05). The pBSilence1.1-cortactin-shRNA3 had the strongest knockdown efficiency compared with pBSilence1.1- cortactin-shRNA1 and pBSilence1.1-cortactin-shRNA2 groups(P＜0.05). Conclusion The constructed and screened shRNA expression plasmid targeting at the cortactin gene could suppress the cortactin mRNA expression in HepG2 cells significantly, which provides an experimental basis to study the effect of cortactin gene silencing on the biological behavior of liver cancer cells by RNA interference.