Benzopyrene (BP) has been shown to be an inducer of apoptosis in some cell types. To date, due to the lack of an appropriate model system, studies of the cellular and biochemical mechanism(s) by which BP induces apoptosis have been focused on Hepa1c1c7 cells. Moreover, the precise relationship between the bioactivation of BP by CYP1A1 or CYP1B1 and the occurrence of cytotoxicity-mediated apoptosis requires further elucidation. In the present study, we showed that BP-induced apoptosis in RL95-2 cells is accompanied by the activation of caspases. In addition, the mitochondrial changes, including the decrease of mitochondrial potential and the release of mitochondrial cytochrome c and Smac/DIABLO into the cytosol, support the suggestion that the mitochondrial pathway is robustly associated with BP-evoked apoptosis. This study showed the involvement of the nuclear translocation of mitochondrial AIF in BP-induced apoptosis of RL95-2 cells. Exposure to BP upregulates AhR, Hsp90, CYP1A1, CYP1B1, and EH significantly, which might be prerequisites for the conversion of BP to BP-7,8-dihydroxy-9,10-epoxide (BPDE). Although both CYP1A1 and CYP1B1 proteins were upregulated significantly by BP, only CYP1A1 exhibited activity. Thus, CYP1A1 is believed to be a central oxidative enzyme that is ultimately required for formation of BPDE from BP in RL95-2 cells. Altogether, our data showed that RL95-2 cells are susceptible to apoptosis by exposure to BP, and that BP-evoked apoptosis is mediated predominantly by the activation of CYP1A1. Here, we suggest that RL95-2 cells are an excellent model for the investigation of xenobiotic mechanisms associated with CYP1A1 as well as CYP1B1.