Objective: To construct tumor necrosis factor receptor-associated factor 6(TRAF6) and its small hair RNA (shRNA) expression vectors, and to assess their function on rat glomerular mesangial cell(GMC). Methods: The full-length TRAF6 CDS (HA tag) or three kinds of shRNAs targeting TRAF6 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1 and pGenesil-1/GFP,respectively. After confirmed by restriction endonuclease digestion and nucleotide sequencing,the recombinant plasmids were transfected into cultured rat GMC through NeonTM transfection system. Western blot assay was then used to confirm the expression of HA-TRAF6 and find out the optimal shRNA against TRAF6 gene. Results: It was verified by restriction endonuclease digestion and nucleotide sequencing that the constructed eukaryotic vectors were all correct. Western blot assay showed that the plasmids of pcDNA3.1-HA-TRAF6 could express HA-TRAF6 and the TRAF6 shRNA-1(shTRAF6-1) was able to silence the target gene effectively. Conclusion: The expression plasmid of TRAF6 gene and its shRNA were constructed successfully, which provides essential experimental tools for studying biological functions of TRAF6 gene in the future.