Primary liver cancers are classified into three types based on their morphology and cytogenetic characteristics hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (CHC). It is often difficult to distinguish these liver tumors. Glypican-3 (GPC3) is serological and histochemical marker of hepatocellular carcinoma. In order to separate these three types of liver cancers, we analyzed the GPC3 expression in 85 liver resection specimens, including 46 HCCs, 28 ICCs and 11 CHCs. GPC3 immunohistochemical staining was used to distinguish HCC from ICC by comparing with the conventional biomarker, alpha-fetoprotein (AFP). The immunostaining of GPC3 was identified in 78.3% (36/46) of HCCs, 60% (9/15) of well differentiated, 88.9% (16/18) of moderately differentiated and 84.6% (11/13) of poorly differentiated HCCs. It was negative in the ICCs. We confirmed that GPC3 expression is specific to HCC component (8/11, 72.7%) but few samples also showed weakly in ICC component (2/11, 18.2%) of CHC sections among 11 cases compared with HCC biomarkers including AFP and hepatocyto paraffin 1 (HepPar1), and ICC biomarkers cytokeratin (CK) 7 and CK19. Three cases in which the macroscopic features resembled ICC did not express GPC3 even in the pathological HCC component. Most (10/11, 91%) of the pathological cholangiocarcinoma components in CHC showed positive staining for CK7 and CK19. The results of this study suggest that GPC3 is a biomarker that is sensitive and specific to HCC component of CHC, and CK7 and CK19 are markers for pathological cholangiocarcinoma component of CHC.