Tumour recurrence and metastases of hepatocellular carcinoma (HCC) after hepatectomy are the major obstacles of long-term survival. The present study investigated the clinicopathological significance of a possible metastasis regulator Six1 in HCC patients who were undergone hepatectomy. Seventy-two pairs of RNA and 103 pairs of protein from tumour and adjacent nontumour liver tissues of HCC patients were examined. About 85 and 60% of HCC tumour tissues were found to overexpress Six1 mRNA and protein, respectively, compared with nontumour liver tissues. No Six1 protein was detected in HCC nontumour liver tissues and normal liver tissues. Increased Six1 protein expression in HCC patients was significantly correlated with pathologic tumour-node-metastasis (pTNM) stage (P=0.002), venous infiltration (P=0.004) and poor overall survival (P=0.0423). We concluded that Six1 is frequently overexpressed in HCC patients and elevated Six1 protein in HCC patients may be an indication of advanced stage and poor overall survival after hepatectomy.