To investigate the expression and significance of glypican-3 (GPC3) in human hepatocellular carcinoma (HCC). DNA chips were used to measure the expression of mRNAs for members of the glypican and syndecan families of heparan sulfate proteoglycans (HSPGs) in normal liver tissue, non-tumor tissues and HCC. GPC3 protein expression was investigated by immunohistochemical staining in the tissues samples and Western blotting in human HCC cell lines. In addition, the levels of GPC3 protein in the blood were determined by ELISA. Only the expression of GPC3 was found to be markedly elevated in HCCs. In the human HCC cell lines, GPC3 expression was consistently observed, and was mainly located in the cell membrane and cytoplasm. Immunohistochemistry showed a tendency for overall staining of the cytoplasm of cells in the liver carcinoma tissues, but the cell membrane was preferentially stained in poorly differentiated HCC when compared with well-differentiated HCC. Moreover, the cell membrane was preferentially stained in metastatic lesions of HCC when compared with primary HCC lesions. Non-tumor tissues and cholangiocellular carcinoma tissues were not stained. In addition, using HepG2 cells, AG490 and piceatannol, which are signal transducer and activator of transcription 3 (STAT3) inhibitors, each increased the amount of GPC3 mRNA expressed. Assay of the circulating levels of GPC3 protein in chronic liver disease and HCC found that serum GPC3 protein levels were significantly elevated in the latter. GPC3 is highly expressed in HCC, and its expression pattern differs according to the degree of cell differentiation. In addition, the expression of GPC3 is regulated by Janus kinase-STAT signaling. GPC3 shows potential as a tumor biomarker for HCC that can be used for molecularly targeted therapy.