The hepatitis B virus X (HBx) protein has many significant roles in hepatocellular carcinoma (HCC). Our previous research demonstrated that mitofusion-2 (Mfn2), a potential tumor suppressor gene in HCC, is a novel direct target of p53 that exerts apoptotic effects via the mitochondrial apoptotic pathway. However, the relationship between HBx and Mfn2 expression in the development of HCC is unknown. We found that HBx had little direct effect on the expression of Mfn2 or p53 in HCC cells not treated with doxorubicin. However, HBx inhibited the upregulation of Mfn2 in HBx-transfected HCC cells simultaneously treated with doxorubicin or cotransfected with p53 plasmid, as evidenced by Western Blot and real-time PCR. Through electrophoretic mobility shift analysis, we confirmed that HBx interfered with the binding event of the p53 protein and the p53 binding site-oligo of the Mfn2 promoter. Moreover, luciferase assays revealed that the activity of the Mfn2 promoter did not increase when transfected with HBx plasmid in doxorubicin-treated HepG2 cells. These results indicate that HBx impacts p53-mediated transcription of Mfn2, providing insight into the negative effect of HBx against p53-dependent chemotherapeutic agents, such as doxorubicin, used in the treatment of HCC. (C) 2012 Elsevier Inc. All rights reserved.