It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3beta (GSK-3beta) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3beta(Ser9) phosphorylation were strongly elevated, indicating that GSK-3beta was inhibited. GSK-3beta phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptor-neutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3beta phosphorylation required PI3-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3beta(Ser9) phosphorylation was controlled by protein kinase C (PKC)zeta because it was blocked by an inhibitory PKCzeta peptide. The blockage of GSK-3beta phosphorylation markedly inhibited glycogen synthesis and decreased beta-catenin expression. In addition, the overexpression of a constitutively active GSK-3beta reduced AP-1-mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3beta. In conclusion, our results identify GSK-3beta as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3beta could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells.