High-mobility group box 1(HMGB1) has been associated with many human cancers, but the role of HMGB1 in hepatocellular carcinoma (HCC) remains unclear. The aim of this study is to investigate the expression of HMGB1 in human HCC with regard to its clinical significance. Twelve cases of normal liver tissues, 34 cases of HCC and the corresponding liver tissue just around the tumor (LAT) were collected. Then, all the samples were subjected to clinicopathologic examination, reverse transcription-polymerase chain reaction (RT-PCR), Western-blot (WB) and immunohistochemical analysis for the expression of HMGB1. The relationships between HMGB1 mRNA expression and clinicopathologic parameters were analyzed. RT-PCR demonstrated that the expression of relative HMGB1 mRNA (HMGB1/GAPDH) was 0.854 ± 0.172; the highest in the tissue of HCC, significantly up-regulated compared with that of 0.527 ± 0.155 in LAT and of 0.405 ± 0.087 in normal liver tissues (P < 0.001). HMGB1 mRNA overexpression was significantly associated with Edmondson stage, TNM stage, vascular invasion and capsule invasion. Western-blot showed the expression of HMGB1 protein in HCC also as the highest among all the groups. Furthermore this overexpression revealed by immunostaining was predominantly localized in the nuclei of HCC; whereas, none of the stains were seen in normal liver cells and only a trace of it was detected in the cytoplasm of LAT cells. Our results suggested the overexpression of HMGB1 might be an important pathogenetic factor in HCC. The mechanisms of HMGB1 in HCC genesis, development and its possible diagnostic and prognostic roles need to be further explored.