Hepatocellular carcinoma (HCC) is a highly malignant tumor and chronic infection with HBV is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we employed two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 HBV-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio=2, p=0.01) in tumor samples, while 158 spots were down-regulated (ratio=-2, p=0.01). Seventy-one gene products were identified among these spots. Members of the heat shock protein 70 and 90 families were simultaneously up-regulated, while metabolism-associated proteins were decreased in HCC samples. The down regulation of mitochondrial and peroxisomal proteins in these results suggested loss of special organelle functions during HCC carcinogenesis. Four metabolic enzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, indicating S-adenosyl-methionine (AdoMet) deficiency in HCC. Two gene products, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and formimidoyltransferase-cyclodeaminase (FTCD) were identified from inversely altered spots. It suggested that different isoforms or post-translational modifications of these two proteins might play different roles in HCC. For the first time, the over expression of hsp70/Hsp90-organizing protein (HOP) and heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) in HCC tissues was confirmed by Western blot and then immunohistochemistry staining in 70 HCC samples, suggesting their potential as protein tumor markers. In summary, we profiled proteome alterations in HCC tissues and these results may provide useful insights for understanding the mechanism involved in the process of HCC carcinogenesis.