To evaluate the cytotoxicity effects of luteolin (LUT) and kaempferol (KAE) via reactive oxygen species (ROS) mediated mitochondrial targeting on hepatocytes obtained from the liver of hepatocellular carcinoma (HCC) rats. In this study, HCC induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF). In the following, rat liver hepatocytes and mitochondria were isolated and tested for every eventual apoptotic and anti-HCC effects of LUT and KAE. The results of MTT assay showed that LUT and KAE were able to induce selective cytotoxicity in hepatocytes of HCC group in a dose- and time-dependent manner. Treatment of mitochondria from hepatocytes of HCC group with LUT and KAE were accompanied by loss of mitochondrial membrane potential (MMP) and mitochondrial swelling and release of cytochrome c (P < 0.001) via reactive oxygen species (ROS) generation before cytotoxicity ensued. LUT and KAE also increased activation of caspase-3 (P < 0.001 and P < 0.01, respectively). Flow-cytometry analysis indicated that the mode of cell death induced by these flavonoids were mostly apoptosis. Importantly, LUT and KAE were nontoxic for healthy hepatocytes and mitochondria. Therefore, we suggest that LUT and KAE are a good candidate for the complementary therapeutic agent against HCC.