Objective：This study aims to investigate the effect of rat nuclear factor-κB（NF-κB）p65 subunit over-expression and its activity changes on the gene promoter activity of interferon regulatory factor-8（IRF-8），and initially screen the possible p65-binding elements within IRF-8 promoter. Methods：To construct the rat wild type p65 over-expression plasmid（pIRES2-p65 WT），complete sequence coding（CDS）of rat p65 was amplified by polymerase chain reaction（PCR） and cloned into pIRES2-EGFP. Then，S535D and S535A mutation was done respectively based on the wild type p65 over-expression plasmid to construct p65 constitutively active mutant（pIRES2-p65 S535D）and p65 dominant negative mutant（pIRES2-p65 S535A）. The potential p65-binding elements within IRF-8 promoter were predicted by using bioinformatics software. Based on the predicted results，luciferase reporter plasmids of full-length（FL）and three truncated IRF-8 gene promoter were constructed，namely pGL3-IRF-8-FL（-1 892~+174 nt），pGL3-IRF-8-1（-1 360~+174 nt），pGL3-IRF-8-2（-752~+174 nt），pGL3-IRF-8-3（-68~+174 nt）. The above-mentioned plasmids were co-transfected into human embryonic kidney 293T（HEK-293T）cells in different groups. Then，the expression level of p65 was detected by Western blot，and the promoter activity of IRF-8 was detected by luciferase experiment to screen the p65-binding elements. Results：It was verified that above-mentioned plasmid was constructed correctly by PCR analysis and nucleotide sequencing. The plasmids of pIRES2-p65 WT，pIRES2-p65 S535D，pIRES2-p65 S535A were respectively transfected into HEK-293T cells together with pGL3-IRF-8-FL. The luciferase results showed that the activity of IRF-8 promoter was markedly increased in response to pIRES2-p65 WT and pIRES2-p65 S535D，especially the later. However，there was no significant change of IRF-8 promoter activity after over-expression of pIRES2-p65 S535A. The plasmids of pGL3-IRF-8-FL or pGL3-IRF-8-1~3 and pIRES2-p65 were co-transfected into HEK-293T cells，and the result displayed that the activity of pGL3-IRF-8-3 was much lower than that of pGL3-IRF-8-FL，pGL3-IRF-8-1 and pGL3-IRF-8-2，indicating that the region of rat IRF-8 promoter（-752~-68 nt） might contain p65-binding elements. Conclusion：Over-expression of wild-type or continuously activated mutant p65 in HEK-293T cells can significantly promote the activity of IRF-8 promoter，and the p65-binding elements in IRF-8 promoter might be located in the -752~-68 nt region.