Objective To explore the cytotoxicity effects of Caspian cobra (Naja oxiana or N. oxiana) venom on hepatocytes and mitochondria obtained from the liver of HCC rats. Methods In this study, HCC was induced by diethylnitrosamine (DEN), as an initiator, and 2-acetylaminofluorene (2-AAF), as a promoter. Rat liver hepatocytes and mitochondria for evaluation of the selective cytotoxic effect of N. oxiana venom were isolated and mitochondria and cellular parameters related to apoptosis signaling were then determined. Results Our results showed a raise in mitochondrial reactive oxygen species (ROS) level, swelling in mitochondria, mitochondrial membrane potential (Δψm) collapse and release of cytochrome c after exposure of mitochondria only isolated from the HCC group with the crude venom of the N. oxiana (12.5, 25, and 50 μg/mL). This crude venom also induced caspase-3 activation (P < 0.001) in the hepatocytes obtained only from the HCC rat liver. Conclusions Based on the over all results, we suggested that N. oxiana may be considered as a promising complementary therapeutic agent for the treatment of HCC.