Common techniques for the treatment of Hepatocellular carcinoma (HCC) have not been successful, and thus the design and discovery of new compounds with better anti-cancer function are needed. Snake venom is among the most important compounds used by researchers to the treatment of various cancers. This study was designed to evaluate the toxicity effect of Persian Gulf snake venom (Enhydrina schistose) on hepatocytes and mitochondria isolated from HCC rats model. HCC has been induced in rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF). The results showed that E. schistose (5, 10, 20 and 40 µg/ml) increases the level of reactive oxygen species (ROS) generation, collapse in mitochondrial membrane potential (MMP), swelling in mitochondria, and cytochrome c release only in hepatocytes and mitochondria isolated from the HCC group. These results proposed that E. schistose could be considered as a promising complementary therapeutic agent for the treatment of HCC.