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The relationship between overexpression of glypican (GPC)-3 that is specific for hepatocellular carcinoma (HCC) and the prognosis has not yet been clarified. We attempted to determine the expression profile of GPC3 in association with the clinicopathological factors by immunohistochemical analysis in HCC patients and investigated the potential prognostic value of GPC3 by comparing the survival rate between the GPC3-positive and GPC3-negative HCC patients. Primary HCC tissue samples (n = 107) obtained from patients who had undergone hepatectomy between 2000 and 2001 were analyzed. GPC3 expression was less frequently observed in well-differentiated HCC than in moderately and poorly differentiated HCC, the difference in the frequency being statistically significant. GPC3-positive HCC patients had a significantly lower 5-year survival rate than the GPC3-negative HCC patients (54.5 vs 87.7%, P = 0.031). Among 80 of the 107 (74.6%) patients with initial treatment who underwent hepatectomy, none of GPC3-negative HCC patients (n = 16, 20.0%) died during the follow-up period. No deaths were noted in the GPC3-negative HCC patients among the 71 (88.7%) patients with moderately and poorly differentiated HCC. Multivariate analysis identified GPC3 expression (P = 0.034) as an independent prognostic factor for the overall survival. We showed that GPC3 expression is correlated with a poor prognosis in HCC patients. (Cancer Sci 2009)