BACKGROUND Oxidative stress is an important factor in the modulation of both tumorigenesis and anticancer responses. Ozone (O3) is a strong oxidant that causes redox reactions and exerts anticancer effects in various types of cancer cells. However, the pathways involved in O3-induced cell death are not well understood. METHODS In vitro human hepatocellular carcinoma (HCC) BEL7402 cells were treated with various O3 concentrations to evaluate O3 cytotoxicity by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The regulatory mechanisms were analyzed by western blot analysis. In vivo, an HCC model was established to evaluate the inhibition of HCC with O3 treatment. RESULTS In vitro cells treated with O3 exhibited a round and small morphology with nuclear shrinkage and fragmentation. The CCK-8 assay confirmed the potent cytotoxic activity of O3 against BEL7402 cells (IC50 value of 5 µg/mL). Acridine orange/ethidium bromide (AO/EB) staining revealed apoptosis of BEL7402 cells after O3 treatment. Flow cytometry analysis showed that S phase cell cycle arrest and apoptosis increased with O3 exposure. In addition, O3 exposure reduced the mitochondrial membrane potential (ΔΨm) and induced reactive oxygen species (ROS) accumulation. Western blot analysis showed that O3 exposure reduced B-cell lymphoma 2 (BCL-2) expression and increased cleaved poly ADP-ribose polymerase (PARP), cytochrome C (Cyt-C), caspase-3, caspase-9, and p-JNK expression. In vivo, treatment with intratumor injection O3 (20 µg/mL) inhibited HCC growth. CONCLUSIONS Overall, our findings showed that O3 induces BEL7402 cell apoptosis via the intrinsic mitochondria-dependent pathway. Therefore, O3 has therapeutic potential for HCC.