Abstract: Objective To construct the HepG2-HBX cell line that stably expressing the hepatitis B virus protein X (HBX) and to explore the specific silencing effect of siRNA on the HBX gene. Methods The pCDNA3.1（+）/HBX-Flag plasmid containing the HBX sequence was transfected into the HepG2 cell lines with lipofectamine 2000, and the HepG2-HBX cell line stably expressing the HBX gene was screened out with G418; RT- PCR and immunohistochemistry were employed to validate the expression of the HBX gene; the siHBX fragment targeting the HBX gene was chemically synthesized and transfected in to the HepG2-HBX cell line, and RT-PCR, real time quantitative RT-PCR and Western blotting were respectively performed to evaluate the the expression of HBX at both the mRNA and protein level in order to validate the specific silencing effect of siHBX on the HBX gene，moreover，the cell cycle of HBX19 was detemined by flow cytometry . Results: The HepG2-HBX cell line stably expressing the HBX gene was successfully constructed and the siHBX fragment could specifically inhibit HBX expression with high efficiency, moreover, it was shown to inhibit cell cycle progression. Conclusions: siHBX could be used as a targeted strategy to inhibit the HBX gene in liver cancer cells, laying an experimental basis for the subsequent therapeutic research in HBV infection-related liver cancers.