Abstract: The aim of this study was to investigate whether melatonin, a free radical scavenger and a general antioxidant, regulates the brain cell apoptosis caused by carbon ions in mice at the level of signal transduction pathway. Young Kun-Ming mice were divided into five groups: control group, irradiation group and three melatonin (1, 5, and 10 mg/kg daily for 5 days i.p.) plus irradiation-treated groups. An acute study was carried out to determine oxidative status, apoptotic cells, and mitochondrial membrane potential (ΔΨm) as well as pro- and anti-apoptotic protein levels in a mouse brain 12 hr after irradiation with a single dose of 4 Gy. In irradiated mice, a significant rise in oxidative stress and apoptosis (TUNEL positive) was accompanied by activated expression of Bax, cytochrome c, caspase-3, and decreased ΔΨm level. Melatonin supplementation was better able to reduce irradiation-induced oxidative damage marked by carbonyl or malondialdehyde content, and stimulate the antioxidant enzyme activities (superoxide dismutase and catalase) together with total antioxidant capacity. Moreover, administration with melatonin pronouncedly elevated the expression of Nrf2 which regulates redox balance and stress. Furthermore, melatonin treatment mitigated apoptotic rate, maintained ΔΨm, diminished cytochrome c release from mitochondria, down-regulated Bax/Bcl-2 ratio and caspase-3 levels, and consequently inhibited the important steps of irradiation-induced activation of mitochondrial pathway of apoptosis. Thus, we propose that the anti-apoptotic action with the alterations in apoptosis regulator provided by melatonin may be responsible at least in part for its antioxidant effect by the abolishing of carbon ion–induced oxidative stress along with increasing Nrf2 expression and antioxidant enzyme activity.