It is well known that telomerase activation and virus infection are strongly associated with human hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) plays an important role in HCC pathogenesis. However, the mechanisms of HBx on telomerase activity are not well understood. To determine the potential roles of HBx in telomerase activity, both transfection and antisense assay were designed to examine the effects of HBx on telomerase in this report. Results showed that HBX gene increased telomerase activity and human telomerase reverse transcriptase (hTERT) expression in HBx-transfected cells and HBx-positive HCC samples. Co-transfection and luciferase reporter assay showed that HBx could stimulate hTERT promoter in a dose-dependent manner in different cells. Truncated and mutant reporter assays revealed that Sp1 binding sites mapped at -132 to +5 nt in hTERT promote were important for HBx-mediated upregulation of hTERT. Western blot did not show any change of Sp1 expression in HBx-transfected cells, but EMSA showed evidence of that HBx increased binding of Sp1 to its target DNA. These results provide new insights into the role of HBx in liver carcinogenesis.