Inappropriate activation of the Wnt/β-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how β-catenin works remains to be elucidated. To identify, in vivo, the target genes of β-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated β-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the β-catenin pathway in the liver. In different mouse models bearing an activated β-catenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked up-regulation of these three genes. The cellular distribution of GS and GLT-1 parallels β-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of β-catenin. In addition, the GS promoter was activated in the liver of GS+/LacZ mice by adenovirus vector-mediated β-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with β-catenin activation. Together, our results indicate that GS is a target of the Wnt/β-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by β-catenin is a contributing factor to liver carcinogenesis.