In spite of carbon ion radiotherapy is a talented modality for malignant tumor patients, the radiation damage of normal tissues adjacent to tumor and the dysfunction of immune system limits therapeutic gain. Protecting immune system against carbon ion radiation-caused damage has the possibility to improve cancer treatment, but it is uncertain whether conventional radioprotective agents play a role in carbon ion radiation. To certify carbon ion caused immune dysfunction and assess the radioprotective effect of melatonin on immune system, animal experiments were performed in radiosensitive BALB/C mice. Here, we observed the bodyweight loss, death and apoptosis, abnormal T-cell distributions in immune system in carbon ion radiated mice. Pretreatment with melatonin could increase the index of thymus and spleen, reduce cell apoptosis in thymus and spleen, and attenuate the carbon ion radiation-caused imbalance of T lymphocytes and disorder of cytokines. These results suggest that melatonin can act as an effective protector against carbon ion radiation-caused immune dysfunction. Furthermore, we also found melatonin restored the activity of the antioxidant enzymes and reduced the level of lipid peroxidation in serum. These data have provided baseline information both for radiation workers and cancer patients to use melatonin as a radioprotector during the carbon ion radiation treatment.