Objective: To construct the eukaryotic expression plasmid carried mouse S100A16 gene short hairpin RNA(shRNA), and to investigate the silencing effect of shRNA on the S100A16 gene in mouse 3T3-L1 cells. Methods:siRNAs were designed according to the S100A16 cDNA sequence in GenBank,and inserted into plasmid PLKO.1-sP6-GFP. The recombinant plasmids were identified by PCR and sequence analysis, and transfected into 3T3-L1 cells with LipofectamineTM 2000. The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by realtime RT-PCR. Results:The correct sequences of recombinant PLKO.1-S100A16-GFP-shRNAs were confirmed by DNA sequencing. Green fluorescence observed by fluorescence microscope showed that shRNAs had been transfected into 3T3-L1 cells(transfection rate was 90%). And the expression of S100A16 mRNA was effectively down-regulated by PLKO.1-S100A16-GFP-shRNA2(inhibition rate was above 70%). Conclusion:The eukaryotic expression plasmids of S100A16 shRNAs were constructed successfully. The PLKO.1-S100A16-GFP-shRNA2 could down-regulated the expression of S100A16 gene effectively. This is fundamental for the study on the function of S100A16 gene.