SS-31, a novel mitochondria-targeted peptide, has been proven to provide neuroprotection in a variety of neurological diseases. Its role as a mitochondrial reactive oxygen species (ROS) scavenger and the underlying pathophysiological mechanisms in traumatic brain injury (TBI) are still not well understood. The aim of the designed study was to investigate the potential neuroprotective effects of SS-31 and fulfill our understanding of the process of the mitochondrial change in the modified Marmarou weight-drop model of TBI. Mice were randomly divided into sham, TBI, TBI + vehicle, and TBI + SS-31 groups in this study. Peptide SS-31 (5 mg/kg) or vehicle was intraperitoneally administrated 30 min after TBI with brain samples harvested 24 h later for further analysis. SS-31 treatment significantly reversed mitochondrial dysfunction and ameliorated secondary brain injury caused by TBI. SS-31 can directly decrease the ROS content, restore the activity of superoxide dismutase (SOD), and decrease the level of malondialdehyde (MDA) and the release of cytochrome c, thus attenuating neurological deficits, brain water content, DNA damage, and neural apoptosis. Moreover, SS-31 restored the expression of SIRT1 and upregulated the nuclear translocation of PGC-1α, which were proved by Western blot and immunohistochemistry. Taken together, these data demonstrate that SS-31 improves the mitochondrial function and provides neuroprotection in mice after TBI potentially through enhanced mitochondrial rebiogenesis. The present study gives us an implication for further clinical research.