Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Southeast Asia and Hong Kong. Among the four proteins that are encoded by the HBV genome, HBV X (HBx) is the most potentially oncogenic factor. It is known that HBx plays an important role in hepatocarcinogenesis, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In this study, we constructed expression vectors for small hairpin RNAs (shRNA) against HBx and investigated their regulatory effects in PLC/PRF/5 HCC cells, which constitutively produce HBx. Our data show that this tool of RNA interference (RNAi) could successfully reduce the HBx mRNA and protein levels by 50-95%. RNAi targeting HBx in PLC/PRF/5 cells demonstrated significant reduction in cell proliferation, cell growth, anchorage-independent growth in soft agar, and tumour development in nude mice. In addition, depletion of HBx expression increased cell sensitivity to TNFalpha-mediated and serum-free-induced apoptosis, and reduced the expression levels of C-myc and Bcl-X(L). These findings suggest that HBx plays an important role in tumorigenicity and anti-apoptotic mechanisms in HCC.