Melatonin, which is a very effective reactive oxygen species (ROS) scavenger, acts through a direct reaction with free radicals. Ca2+ entry induced by traumatic brain injury (TBI) has deleterious effects on human hippocampal function. TRPM2 is a Ca2+ permeable non-selective channel in hippocampal neurons, and its activation of during oxidative stress has been linked to cell death. Despite the importance of oxidative stress in TBI, its role in apoptosis and Ca2+ entry in TBI is poorly understood. Therefore, we tested the effects of melatonin on apoptosis, oxidative stress, and Ca2+ entry through the TRPM2 channel in the hippocampal neurons of TBI-induced rats. Thirty-two rats were divided into the following four groups: control, melatonin, TBI, and TBI + melatonin groups. Melatonin (5 mg/kg body weight) was intraperitoneally given to animals in the melatonin group and the TBI + melatonin group after 1 h of brain trauma. Hippocampal neurons were freshly isolated from the four groups, incubated with a nonspecific TRPM2 blocker (2-aminoethyl diphenylborinate, 2-APB), and then stimulated with cumene hydroperoxide. Apoptosis, caspase-3, caspase-9, intracellular ROS production, mitochondrial membrane depolarization and intracellular free Ca2+ ([Ca2+](i)) values were high in the TBI group, and low in the TBI + melatonin group. The [Ca2+](i) concentration was decreased in the four groups by 2-APB. In our TBI experimental model, TRPM2 channels were involved in Ca2+ entry-induced neuronal death, and the negative modulation of the activity of this channel by melatonin pretreatment may account for the neuroprotective activity of TRPM2 channels against oxidative stress, apoptosis, and Ca2+ entry.