Objective To construct five shRNA-expression plasmids and to investigate the expression of EM>Smad2/EM> in TGFβ/ Smads signal transduction treated with shRNA-expression plasmid. Methods Five shRNA-EM>Smad2/EM> DNA sequences from mRNA sequence of mouse EM>Smad2/EM> gene were designed and synthesized. DNA oligonucleotides encoding an appropriate shRNA were inserted to shRNA expression vector respectively. Five shRNAEM>Smad2/EM> expression plasmids were obtained and then transfected into NIH/3T3 cells. The suppressed expression of EM>Smad2/EM> was assessed by RT-PCR and Westernblotting. Results The shRNA-expression plasmid numbered 2.4 could markedly reduce the expression of EM>Smad2/EM>. The suppression effect of the RNAipool composed of four different plasmids was more obvious than that of any single. Conclusion The shRNAexpression plasmids were successfully constructed, which could specifically and effectively suppress the expression of EM>Smad2