Sarcomas are relatively resistant because of hypoxia. We previously demonstrated that the transcutaneous CO2 therapy reduced hypoxic conditions in human malignant fibrous histiocytoma (MFH). Therefore, we hypothesized that transcutaneous CO2 therapy could enhance the antitumor effect of radiation therapy in human MFH. Our purpose was to evaluate the effects of transcutaneous CO2 therapy on the antitumor efficacy of X-ray irradiation using MFH. First, in an in vitro study, we assessed apoptotic activity and reactive oxygen species (ROS) production using flow cytometric and immunoblot analysis at 24 h after X-ray irradiation under three different oxygen conditions (normoxic, reoxygenated and hypoxic). In addition, in the in vivo study, 24 male athymic BALB/c nude mice with MFH tumors that were inoculated in the dorsal subcutaneous area were randomized into four groups: control, CO2, X-ray irradiation and combination (CO2 and X-ray irradiation). Treatments were performed twice weekly for 2 weeks, four times in total. Tumor volume was calculated. All tumors were excised and apoptotic activity, ROS production, related proteins and HIF-1α expression were assessed using flow cytometric and immunoblot analysis. The in vitro study revealed that X-ray irradiation induced increased apoptosis and ROS production in MFH cells under normoxic and reoxygenated conditions relative to hypoxic conditions (P<0.01). In the in vivo study, tumor volume in the combination group was reduced to 28, 42 and 47% of that in the control, CO2, and X-ray groups, respectively (P<0.05). Apoptotic activity and ROS production in the combination group were strongly increased with decreasing HIF-1α expression relative to the control, CO2 and X-ray groups. The transcutaneous CO2 system enhanced the antitumor action of X-ray irradiation and could be a novel therapeutic tool for overcoming radioresistance in human malignancies.