Objective Scorpion(Hemiscorpius lepturus) stings are a public health concern in Iran, particularly in south and southwestern regions of Iran. The gold standard for the treatment of a scorpion sting is anti-venom therapy. However, immunotherapy can have serious side effects, such as anaphylactic shock(which can sometimes even lead to death). The aim of the current study was to demonstrate the protective effect of ozone against toxicity induced by Hemiscorpius lepturus(H. lepturus) venom in mice. Methods Eight hours after the injection of ozone to the experimental design groups, the male mice were decapitated and mitochondria were isolated from five different tissues(liver, kidney, heart, brain, and spinal cord) using differential ultracentrifugation. Then, assessment of mitochondrial parameters including mitochondrial reactive oxidative species(ROS) production, mitochondrial membrane potential(MMP), ATP level, and the release of cytochrome c from the mitochondria was performed. Results Our results showed that H. lepturus venom-induced oxidative stress is related to ROS production and MMP collapse, which is correlated with cytochrome c release and ATP depletion, indicating the predisposition to the cell death signaling. Conclusion In general, ozone therapy in moderate dose can be considered as clinically effective for the treatment of H. lepturus sting as a protective and antioxidant agent. Objectives Scorpion (Hemiscorpius lepturus) stings are a public health concern in Iran, particularly in south and southwestern regions of Iran. However, the gold standard for the treatment of a scorpion sting is anti-venom therapy. However, immunotherapy can have serious side effects, such The aim of the current study was to demonstrate the protective effect of ozone against toxicity induced by Hemiscorpius lepturus (H. lepturus) venom in mice. Methods Eight hours after the injection of ozone to the experimental design groups, the male mice were decapitated and mitochondria were isolated from five different tissues (liver, kidney, heart, brain, and spinal cord) using differential ultracentrifugation. Then, assessment of mitochondrial parameters including mitochondrial reactive oxidative species (ROS) production, mitochondrial membrane potential (MMP), ATP level, and the release of cytochrome c from the mitochondria was performed. Our results showed that H. lepturus venom-induced oxidative stress is related to ROS production and MMP collapse, which is correlated with cytochrome c release and ATP depletion, indicating the predisposition to the cell death signaling. Conclusion In general, ozone therapy in moderate dose can be considered as clinically effective for the treatment of H. lepturus sting as a protective and antioxidant agent.