In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG₂ to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG₂ cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG₂/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG₂/GFP-Daxx cells). After incubation with hydrogen peroxide (H₂O₂) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG₂/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG₂ cells and observed that the hydrogen peroxide decreased the viability of HepG₂ cells in concentration-dependent pattern. The IC₅₀ values in three groups (Normal cells, HepG₂/GFP cells and HepG₂/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG₂/GFP-Daxx cells as compared to the other two groups. HepG₂/GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG₂ cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.