In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5‐Aza‐dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor‐2 (TFPI‐2), a Kunitz‐type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI‐2 was frequently silenced in human HCC and HCC cell lines. TFPI‐2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI‐2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI‐2 messenger RNA expression. In addition, TFPI‐2 expression in HCC cell lines can be robustly restored by combined treatment with 5‐Aza‐dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI‐2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI‐2 significantly suppressed the proliferation and invasiveness of HCC cells. Conclusion: Our findings suggest that TFPI‐2 is a candidate tumor suppressor gene in human HCC. (HEPATOLOGY 2007;45:1129–1138.)