Melatonin has been proven to possess neuroprotection property against various neurological diseases by decreasing cerebral oxidative stress and inhibiting inflammatory process. However, whether administration of melatonin influences the autophagy pathway, which has recently been reported playing a pivotal role in traumatic brain injury, is yet not fully understood. We supposed that treatment of melatonin enhances the autophagy pathway after traumatic brain injury (TBI) in mice and subsequently inhibited the mitochondrion apoptotic pathway. Firstly, we investigated the neurological severity score, brain water content and neuronal apoptosis in mice cortex to demonstrate the neuroprotection of melatonin. Then we determined the autophagy markers, namely Beclin1 and LC3-II, using western blot and immunofluorescence. Next, we evaluated the mitochondrial apoptotic pathway in the presence or absence of melatonin. More significantly, we employed 3-methyladenine (3-MA) to inhibit the autophagy pathway, to further confirm our hypothesis. The results showed that melatonin significantly ameliorated secondary brain injury induced by TBI. In addition, melatonin enhanced autophagy after TBI, which was accompanied by a decrease in both the translocation of Bax to mitochondria and the release of cytochrome C to cytoplasm. Furthermore, simultaneous treatment of 3-MA reversed the beneficial effects of melatonin on mitochondrial apoptotic pathway. Taken together, we conclude that melatonin enhances autophagy, which inhibits mitochondrial apoptotic pathway, thus protecting mice from secondary brain injury after TBI.