The ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region-containing protein (ASPP) family of proteins regulates apoptosis through interaction with p53 and its family members. This study evaluated the epigenetic regulation of ASPP1 and ASPP2 in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC) and explores the effects of down-regulation of ASPP1 and ASPP2 on the development of HCC. HCC cell lines and tissues from HCC patients were used to examine the expression and methylation of ASPP1 and ASPP2. The expression of ASPP1 and ASPP2 was diminished in HCC cells by epigenetic silence owing to hypermethylation of ASPP1 and ASPP2 promoters. Analyses of 51 paired HCC and surrounding nontumor tissues revealed that methylation of ASPP1 and ASPP2 was associated with the decreased expression of ASPP1 and ASPP2 in tumor tissues and the early development of HCC. Moreover, ASPP2 became methylated upon HBV x protein (HBx) expression. The suppressive effects on tumor growth by ASPP1 and ASPP2 were examined with RNA interference-mediated gene silence. Down-regulation of ASPP1 and ASPP2 promoted the growth of HCC cells in soft agar and in nude mice and decreased the sensitivity of HCC cells to apoptotic stimuli. Conclusion: ASPP1 and ASPP2 genes are frequently down-regulated by DNA methylation in HBV-positive HCC, which may play important roles in the development of HCC. These findings provide new insight into the molecular mechanisms leading to hepatocarcinogenesis and may have potent therapeutic applications. Copyright © 2009 by the American Association for the Study of Liver Diseases.