Perturbations to the Wnt signaling pathway have been implicated in a large proportion of human hepatocellular carcinomas (HCCs). Activating β-catenin mutations and loss of function mutations in Axin1 are thought to be functionally equivalent. We examined the Wnt pathway in HCC by comparing the expression of β-catenin target genes and the level of β-catenin-dependent transcriptional activation, in 45 HCC tumors and four cell lines. Among these samples, β-catenin and AXIN1 were mutated in 20 and seven cases, respectively. We found a significant correlation between activated β-catenin mutations and overexpression of mRNA for the target genes glutamine synthetase (GS), G-protein-coupled receptor (GPR)49 and glutamate transporter (GLT)-1 (P = 0.0001), but not for the genes ornithine aminotransferase, LECT2, c-myc and cyclin D1. We also showed that GS is a good immunohistochemical marker of β-catenin activation in HCC. However, we observed no induction of GS, GPR49 or GLT-1 in the five inactivated Axin1 tumors. β-Catenin-dependent transcriptional activation in two Axin1-mutated HCC cell lines was much weaker than in β-catenin-mutated cell lines. Our results strongly suggest that in HCC, contrary to expectation, the loss of function of Axin1 is not equivalent to the gain of function of β-catenin. Our results also suggest that the tumor suppressor function of Axin1 in HCC may be related to another, non-Wnt pathway.