Objective:To construct shRNA expression vectors targeting of rat ATF3 gene specifically. Methods:Five 19~21 bp shRNAs targeting of rat ATF3 gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1. After being digested by restriction endonuclease and sequencing confirmed,the recombinant plasmids were transfected into rat glomerular mesangial cells(GMC),and then the level of ATF3 protein was measured by Western blot to select the optimal shRNA. Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the eukaryotic vectors expressing shRNA targeting of rat ATF3 gene were successfully constructed. The most optimal shRNA which could effectively silence the target genes was shATF3-1. Conclusion:The eukaryotic vector with the ability of specifically knocking down rat ATF3 gene was constructed successfully.