Osteosarcoma is the most common primary solid malignant bone tumor. Despite substantial improvements in surgery and chemotherapy, metastasis remains a major cause of fatal outcomes, and the molecular mechanisms of metastasis are still poorly understood. Hypoxia, which is common in malignant tumors including osteosarcoma, increases expressions of hypoxia inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2 and MMP-9, and can induce invasiveness. As we previously showed a novel transcutaneous CO2 application to decrease HIF-1α expression and induce apoptosis in malignant fibrous histiocytoma, we hypothesize that transcutaneous CO2 application could suppress metastatic potential of osteosarcoma by improving hypoxic conditions. Here, we examined the effects of transcutaneous CO2 application on apoptosis, and development of pulmonary metastasis using a highly metastatic osteosarcoma cell line, LM8. Transcutaneous CO2 application significantly decreased tumor growth and pulmonary metastasis in LM8 cells. Apoptotic activity increased, and intratumoral hypoxia was improved with decreased expressions of HIF-1α, MMP-2 and MMP-9, significantly, in the CO2-treated tumors. In conclusion, we found that transcutaneous CO2 application can induce tumor cell apoptosis and might suppress pulmonary metastasis by improvement of hypoxic conditions with decreased expressions of HIF-1α and MMPs in highly metastatic osteosarcoma cell. These findings strongly indicate that this novel transcutaneous CO2 therapy could be a therapeutic breakthrough for osteosarcoma patients.