Flumequine (FL), a quinolone antibiotic used for veterinary treatment of bacterial infections, has been reported to exert hepatocarcinogenicity in mice, and oxidative stress has been considered as a possible underlying mechanism. To elucidate the tumor-promoting mechanism of FL in mice, we employed a two-stage liver carcinogenesis model. Livers from mice were subjected to histopathological examinations, histochemistry for γ-glutamyltranspeptidase (GGT), and immunohistochemistry for PCNA. In addition, gene expression analyses were performed using low-density cDNA microarrays for mouse stress and toxicity and drug metabolism, and quantitative real-time RT-PCR analyses. On histopathological examinations, centrilobular hypertrophy, vacuolation, and mitotic figures of liver cells were observed in animals receiving FL, with or without DEN-initiation. The number of GGT- and PCNA-positive hepatocytes in the DEN+FL group was significantly higher than that in the DEN alone group. In the gene expression analysis, animals of the DEN+FL group showed significant up-regulations of oxidative and metabolic stress related genes, such as glutathione S-transferase (Gst) α2, Gstμ2, microsomal epoxide hydrolase (Ephx1), NAD(P)H:quinone oxidoreductase 1 (Nqo1), γ-glutamylcysteine synthetase heavy subunit (γGCSh), and NF-E2 related factor 2 (Nrf2), as well as extracellular signal regulated kinase 5 (ERK5) and c-Jun, as compared to the DEN alone group. Additionally, the in vitro measurement of reactive oxygen species (ROS) generated in the mouse liver microsomes showed a significant increase of ROS production induced by FL. These results support our previous hypothesis that oxidative stress plays an important role in FL-induced hepatocarcinogenesis in mice.