Recent studies have shown that intratumoral cholestasis is a hallmark of CTNNB1 mutations in hepatocellular carcinomas (HCC). Here, we analyzed the expressions of genes involved in bile acid and bilirubin metabolism and their correlation with the mutational status of CTNNB1 in a series of HCC. The expressions of CYP7A1 and CYP27A1, which encode rate-limiting enzymes in bile acid synthesis, were unaltered or only marginally increased in CTNNB1-mutated HCC compared with those in HCC with wild-type CTNNB1. Among the genes involved in bile acid and bilirubin transport, the expression of SLCO1B3 was significantly elevated in HCC with CTNNB1 mutations, whereas the expression of ABCC4 was elevated in HCC with wild-type CTNNB1. Immunohistochemistry confirmed the frequent expression of SLCO1B3 in CTNNB1-mutated HCC at the protein level, but not in most HCC with wild-type CTNNB1. Immunohistochemistry for MRP4 (encoded by ABCC4) partly agreed with ABCC4 expression, but most cases did not express detectable levels of MRP4. Notably, all HCC with bile accumulation, including those without CTNNB1 mutations, expressed SLCO1B3, suggesting that SLCO1B3 expression, rather than CTNNB1 mutation, is the critical determinant of intratumoral cholestasis. As SLCO1B3 is involved in the uptake of a number of chemotherapeutic and diagnostic agents, SLCO1B3 expression and the status of CTNNB1 mutation might need to be considered in the drug delivery to HCC. (Cancer Sci 2011; 102: 1742–1747)