Carbon dioxide (CO2) therapy can be applied to treat a variety of disorders. We previously found that transcutaneous application of CO2 with a hydrogel decreased the tumor volume of several types of tumors and induced apoptosis via the mitochondrial pathway. However, only one condition of treatment intensity has been tested. For widespread application in clinical antitumor therapy, the conditions must be optimized. In the present study, we investigated the relationship between the duration, frequency, and treatment interval of transcutaneous CO2 application and antitumor effects in murine xenograft models. Murine xenograft models of three types of human tumors (breast cancer, osteosarcoma, and malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma) were used to assess the antitumor effects of transcutaneous CO2 application of varying durations, frequencies, and treatment intervals. In all human tumor xenografts, apoptosis was significantly induced by CO2 treatment for ≥10 min, and a significant decrease in tumor volume was observed with CO2 treatments of >5 min. The effect on tumor volume was not dependent on the frequency of CO2 application, i.e., twice or five times per week. However, treatment using 3- and 4-day intervals was more effective at decreasing tumor volume than treatment using 2- and 5-day intervals. The optimal conditions of transcutaneous CO2 application to obtain the best antitumor effect in various tumors were as follows: greater than 10 min per application, twice per week, with 3- and 4-day intervals, and application to the site of the tumor. The results suggest that this novel transcutaneous CO2 application might be useful to treat primary tumors, while mitigating some side effects, and therefore could be safe for clinical trials.