Objective To construct effective short hairpin(shRNA) recombinant plasmids targeting mouse high mobility group box-1 protein(HMGB1) gene. Methods Four pairs of shRNA sequences targeting mouse HMGB1 gene and one pair of negative control shRNA sequence were designed and synthesized.The recombinant plasmids were constructed and identified by Xho I restriction enzyme. Then the four plasmids were transfected into mouse hepatoma cell line Hepa1- 6 for sh RNA screening.H MGB1 knockdown efficiency was evaluated by real-time fluorescent quantitative polymerase chain reaction(FQ- PCR) detecting system analysis. Results The four shRNA recombinant plasmids were constructed correctly. The shRNA screening experiment identified that the fluorescence expression rate of p Yr-1.1-mus HMGB1-sh1-4 and NC is 85%, 78%,75%,70% and 60%; FQ- PCR shows relative expression levels of p Yr-1.1-mus HMGB1-sh1-4 and NCis 0.52±0.05, 0.59±0.08,0.61±0.08, 0.57±0.21 and 1.02±0.21 respectively. Conclusion Four recombinant plasmid of HMGB1-targeted shRNA is successfully constructed and screened, in which p Yr-1.1-mus HMGB1-sh1 exerted the most significant inhibition effect on HMGB1 gene, providing the basis for studying on HMGB1 function. Key words: High-mobility group protein 1; RNA interference; Short hairpin vector