The p53 mutation and altered Pten expression are two most common genetic events in Hepatitis B virus (HBV) infection related hepatocellular carcinoma (HCC). To confirm the causative role of p53 and Pten somatic mutation in HCC development, we established CRISPR/Cas9-mediated somatic gene disruption via hydrodynamic tail vein injection, allowing for in vivo targeting p53 and Pten simultaneously in adult HBV transgenic mice. Here we demonstrated that the utility of this approach resulted in macroscopic liver tumors as early as 4 months' post injection and most tumors harbored both p53 and Pten loss-of-function alterations. Immunohistochemical (IHC) and histopathology analysis demonstrated that the tumors were positive for Glutamine synthetase (GS), a marker of HCC and accompanied with prominent lipid accumulation. The study here indicated that CRISPR/Cas9-mediated p53 and Pten somatic mutation accelerated hepatocarcinogenesis in adult HBV transgenic mice. This method also provides a fast and convenient system for generating mouse model of HCC with HBV infection characteristics.