Objective: To investigate the key factor(s) of hepatitis B virus X protein (HBx) promoting B7-H6 gene activation. Methods: The DNA fragments of the B7-H6 promoter were amplified from the human genomic DNA using polymerase chain reaction(PCR). Products of PCR were digested by KpnI and HindⅢ, and inserted into luciferase reporter vector (pGL3-Basic). The correctness of the recombinant plasmid pGL3-B7-H6 was confirmed by sequencing. Human hepatoma cell line HepG2 were co-transfected with pGL3-B7-H6 and the eukaryotic expression vectors (pCMV-HBs、pCMV-HBc and pCMV-HBx), and the relative luciferase activity was detected.The different dose of HBx expression plasmids were transfected into the HepG2 cells, and the expression of B7-H6 protein were determined by Western blot. Results: A period of 2.2 Kb of B7-H6 promoter region were successfully cloned into pGL3-Basic, which was confirmed by DNA sequencing. The relative luciferase activity was significantly higher in the cells transfected with pGL3-B7-H6 than that in the cells transfected with the empty vector pGL3-Basic (5.24±1.25 vs. 1.12±0.31, P=0.005). The relative luciferase activity in HepG2 cells co-transfected with pGL3-B7-H6 and pCMV-HBx was remarkably increased compared with the control group (17.60±3.36 vs. 6.73±1.36, P=0.001). Western blot further demonstrated that HBx protein can significantly enhance B7-H6 protein expression. Conclusions Hepatitis B Virus X proteins enhanced B7-H6 promoter activity and promoted B7-H6 protein expression.