Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Thus, alternative therapeutic strategies need to be established. In order to search for a useful biomarker to improve its efficacy, we conducted a two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis to profile the differentially expressed proteins between HCC tumor tissues with histological evidence and the adjacent non-tumor tissues. Twenty-two out of 43 dysregulated proteins were identified, including 15 upregulated proteins, and 7 downregulated proteins (over 2-fold, P<0.01). The expression of peroxiredoxin 3 (PRDX3) at the mRNA and protein levels was confirmed by RT-PCR and western blotting in HCC cell lines, and HCC samples, and further analysed by immunohistochemistry in HCC samples of different clinical pathological stages. The results indicated that overexpression of PRDX3 was associated with 94.9% HCC, and correlated with poor differentiation (P<0.05), which suggest that PRDX3 has substantial clinical impact on the progression of hepatocarcinoma, and may be a potential therapeutic target for HCC.