Ezrin phosphorylation is directly involved in regulation of Hippo pathway, Yap levels and growth rates of normal and neoplastic hepatocytes. It also has been shown that coordinated activation of Akt and Yap induces intrahepatic cholangiocarcinoma (ICC) in mice. In this study, we aimed to illustrate if surrogate maker of Yap, p-Ezrin(Thr567) could work with Akt to establish cholangiocarcinogenesis, and its potential translational significance. Initially activated Akt hydrodynamic tail vein injection mouse model was established and studied. Following that, we generated two mouse models by the same approach with mutant Ezrin (T567D) (which mimics p-Ezrin(Thr567)) or wild type Ezrin, in association with or without activated form of Akt. After 3 months, while forced co-expression of wild type Ezrin and Akt in the C57 mouse strain did not induce any appreciable abnormality, mutant Ezrin/Akt mouse model was associated with development of macroscopically visible tumors in the liver. After 5 months, wild type Ezrin and Akt in the C57 mouse strain induced macroscopically visible tumors in the liver. Surprisingly, over expression activated Akt alone in the FVB mouse strain induced macroscopically visible tumor only after 21 weeks of injection. An activated Akt associated “balloon cell” like hepatocytes phenomenon was observed and oil red staining was positive. By H&E staining, these tumor cells in the C57 mouse strain had the histology of purely cholangiocyte neoplasms. However, the countparts in the FVB mouse showed a mixed cholangiocyte and hepatocyte derivation, mixed tumor types, including hepatic adenoma, hepatocellular carcinoma and cholangiocarcinoma, and mixed stages of variant neoplasms. In addition, an intense infiltrating immune cell response along the edge of the neoplasms was observed specifically in the mutant Ezrin/Akt FVB mouse model at all the time points (3 month, 4 month, and 5 month) with a positive Ezrin staining. All those malignant tumor cells were consistently positive for mutant Ezrin (T567D)(GFP), and Akt (HA tag) positive. We also observed Yap, CK19 and PCNA positive staining specifically on these tumor cells. ICC tissue microarray revealed only 24% patients were p-Ezrin(Thr567) positive, this positivity correlated with lower TNM stage, lower tumor grade, and higher Yap expression. In addition, cBioPortal database showed there were total 5.3% ICC patients tested FAT1 and NF2 genomic abnormalities, and 10% ICC patients tested PIK3CA and PTEN genomic abnormalities. Conclusions We successfully established a purely ICC mouse model and a mixed HCC/ICC mouse model, which could be clearly identified macroscopically and microscopically. The fact that these neoplasms arose after hydrodynamic injection, strongly suggests that the cholangiocarcinomas observed arose from hepatocyte to cholangiocyte transdifferentiation. The finding has mechanistic and potentially therapeutic applications in understanding and regulating and trans-differentiation of hepatocytes, growth of cholangiocytes and Intrahepatic Cholangiocarcinoma (ICC) pathogenesis.