The nuclear receptor constitutive active/androstane receptor (CAR) acts as a sensor of toxic byproducts derived from the endogenous metabolism and exogenous chemicals. We previously reported that CAR is responsible for exacerbating hepatic injury and fibrosis in a dietary model of non-alcoholic steatohepatitis (NASH) via upregulation of lipid peroxidation. In this study, we investigated the pathological roles of the CAR in the development of hepatocellular carcinoma in NASH model. CAR+/+ and CAR-/- mice were fed methionine- and choline-deficient (MCD) diet after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine (DEN) at 2 weeks of age. Interestingly, the MCD diet dramatically promoted DEN-induced hepatocarcinogenesis in CAR+/+ mice. However, the deletion of CAR leads to a significantly lower tumor incidence and smaller tumor diameter. Hepatocytes of MCD-treated-CAR+/+ mice showed a significantly higher staining frequency of Ki-67, a marker of cell proliferation, and exhibited a higher expression of c-Myc and FoxM1 transcripts compared with MCD-treated CAR-/- mice. Immunohistochemistry revealed the nuclear translocation of CAR thus suggesting that the activation of CAR signaling increased in the hepatocytes of CAR+/+ mice fed MCD diet. In addition, in vitro experiments using the CAR stably expressed cell line with TCPOBOP have suggested that CAR activation directly leads to cell proliferation. Survival was significantly lower in the CAR+/+ mice fed the MCD diet in comparison with the CAR-/- mice. Taken together, these results suggest that CAR may therefore play a critical role in the hepatocarcinogenesis of the murine NASH model via the upregulation of cell proliferation.