Opisthorchis viverrini (Ov) infection is the major etiological factor for cholangiocarcinoma (CCA), especiallyin northeast Thailand. We have previously reported significant involvement of PI3K/AKT/PTEN and Wnt/β-catenin in human CCA tissues. The present study, therefore, examined the expression and activation of PI3K/AKT/PTEN and Wnt/β-catenin signaling components during Ov-induced cholangiocarcinogenesis in a hamsteranimal model. Hamsters were divided into two groups; non-treated and Ov plus NDMA treated. The resultsof immunohistochemical staining showed an upregulation of PI3K/AKT signaling as determined by elevatedexpression of the p85α-regulatory and p110α-catalytic subunits of PI3K as well as increased expression andactivation of AKT during cholangiocarcinogenesis. Interestingly, the staining intensity of activated AKT (p-AKT)increased in the apical regions of the bile ducts and strong staining was detected where the liver fluke resides.Moreover, PTEN, a negative regulator of PI3K/AKT, was suppressed by decreased expression and increasedphosphorylation during cholangiocarcinogenesis. We also detected upregulation of Wnt/β-catenin signaling asdetermined by increased positive staining of Wnt3, Wnt3a, Wnt5a, Wnt7b and β-catenin, corresponded withthe period of cholangiocarcinogenesis. Furthermore, nuclear staining of β-catenin was observed in CCA tissues.Our results suggest the liver fluke infection causes chronic inflammatory conditions which lead to upregulationof the PI3K/AKT and Wnt/β-catenin signaling pathways which may drive CCA carcinogenesis. These resultsprovide useful information for drug development, prevention and treatment of CCA.