OBJECTIVE To construct an eukaryotic expression plasmid of human TNF receptor-associated factor 3 in teracting protein 3(TRAF3IP3) gene and identify its expression in HEK293 cells. METHODS Human TRAF3IP3 cDNA was amplified by RT-PCR from bone marrow mononuclear cells. After digested by restriction enzymes XhoI and SalI, the complete open reading frame of TRAF3IP3 gene was inserted into pIRES2-EGFP eukaryotic expression vector with a Flag tag at the N-terminus. The recombinant plasmid was identified by double restriction enzyme digestion and sequencing analysis. The constructed TRAF3IP3 eukaryotic expression plasmid was transfected into HEK293 cells by calcium phosphate precipitation method. The expression of green fluorescence protein was observed by fluorescence microscopy. Real-time PCR and Western blotting were performed to detect the expression of Flag-TRAF3IP3 fusion protein. RESULTS Double restriction enzyme digestion and sequencing analysis revealed that TRAF3IP3 eukaryotic expression plasmid was constructed successfully. Green fluorescence was detected in transfected HEK293 cells. Real-time PCR showed TRAF3IP3 mRNA was expressed at a high level. The approximate 62 kD Flag-TRAF3IP3 fusion protein was found by Western blotting. CONCLUSIONS Human TRAF3IP3 eukaryotic expression plasmid pIRES2-EGFP-TRAF3IP3 has been constructed successfully, which provides a foundation for the gene function research of TRAF3IP3.