Objective: To compare the transfection efficiency and cytotoxicity among Lipo2000 reagent, calcium phosphate method, and Lonza electroporation in transfection of hepatitis B virus X (HBx) gene expression plasmid (pHA-HBx) into the HepG2 cells. Methods: Green fluorescent protein (GFP)-tagged pHA-HBx was transfected into the HepG2 cells by Lipo2000 reagent, calcium phosphate method, and Lonza electroporation-based delivery, respectively. Then, the transfection efficiency was determined by flow cytometry and fluorescence microscopy, the HBx gene integration into the genome of the HepG2 cells was identified by RT-PCR method, and the cell survival status was examined and analyzed by Annexin V/PI staining, respectively. Results: RT-PCR results indicated that exogenous HBx gene was integrated into the genome and expressed in the HepG2 cells after either of the three transfection methods. The transfection efficiency of Lipo2000 reagent and Lonza electroporation-based delivery were higher than that of calcium phosphate method (both P<0.05), but it had no statistical difference between the first two methods (P>0.05). The high-to-low order of cell survival rate was Lonza electroporation, Lipo2000 reagent and calcium phosphate method, respectively (all P<0.05). Conclusion: Lonza electroporation for pHA-HBx transfection into HepG2 cells has high transfection efficiency and less cytotoxicity, so it is a relatively satisfactory transfection method.