Peroxisome proliferators, such as lipid-lowering fibrate drugs, are agonists for the peroxisome proliferator-activated receptor α (PPARα). Sustained activation of PPARα leads to the development of liver tumors in rodents. Paradoxically, humans appear to be resistant to the induction of peroxisome proliferation and development of liver tumors by peroxisome proliferators. To examine the species differences in response to peroxisome proliferators, a PPARα humanized mouse (hPPARα) was generated, in which the human PPARα was expressed in liver under control of the Tet-OFF system. To evaluate the susceptibility of hPPARα mice to peroxisome proliferator-induced hepatocarcinogenesis, a long-term feeding study of Wy-14,643 was carried out. hPPARα and wild-type (mPPARα) mice were fed either a control diet or one containing 0.1% Wy-14,643 for 44 and 38 weeks, respectively. Gene expression analysis for peroxisomal and mitochondrial fatty acid metabolizing enzymes revealed that both hPPARα and mPPARα were functional. However, the incidence of liver tumors including hepatocellular carcinoma was 71% in Wy-14,643-treated mPPARα mice, and 5% in Wy-14,643-treated hPPARα mice. Upregulation of cell cycle regulated genes such as cd1 and Cdks were observed in non-tumorous liver tissue of Wy-14,643-treated mPPARα mice, whereas p53 gene expression was increased only in the livers of Wy-14,643-treated hPPARα mice. These findings suggest that structural differences between human and mouse PPARα are responsible for the differential susceptibility to the peroxisome proliferator-induced hepatocarcinogenesis. This mouse model will be useful for human cancer risk assessment of PPARα ligands.