The purpose of the present study was to establish a non-surgical breast-conserving treatment (BCT) using KORTUC II radiosensitization treatment. A new radiosensitizing agent containing 0.5% hydrogen peroxide and 0.83% sodium hyaluronate (a CD44 ligand) has been developed for intra-tumoral injection into various tumors. This new method, named KORTUC II, was approved by our local ethics committee for the treatment of breast cancer and metastatic lymph nodes. A total of 72 early-stage breast cancer patients (stage 0, 1 patient; stage I, 23; stage II, 48) were enrolled in the KORTUC II trial after providing fully informed consent. The mean age of the patients was 59.7 years. A maximum of 6 mL (usually 3 mL for tumors of less than approximately 3 cm in diameter) of the agent was injected into breast tumor tissue twice a week under ultrasonographic guidance. For radiotherapy, hypofraction radiotherapy was administered using a tangential fields approach including an ipsilateral axillary region and field-in-field method; the energy level was 4 MV, and the total radiation dose was 44 Gy administered as 2.75 Gy/fraction. An electron boost of 3 Gy was added three times. Treatment was well tolerated with minimal adverse effects in all 72 patients. No patients showed any significant complications other than mild dermatitis. A total of 24 patients under 75 years old with stage II breast cancer underwent induction chemotherapy (EC and/or taxane) prior to KORTUC II treatment, and 58 patients with estrogen receptor-positive tumors also received hormonal therapy following KORTUC II. The mean duration of follow-up as of the end of September 2014 was 51.1 months, at which time 68 patients were alive without any distant metastases. Only one patient had local recurrence and died of cardiac failure at 6.5 years. Another one patient had bone metastases. For two of the 72 patients, follow-up ended after several months following KORTUC II treatment. In conclusion, non-surgical BCT can be performed using KORTUC II, which has three major characteristics: imaging guidance; enzyme-targeting; and targeting of breast cancer stem cells via the CD44 receptor.