Traumatic brain injury (TBI) is a major cause of disability and death, and a better understanding of the underlying mechanisms of mitochondrial dysfunction will provide important targets for preventing damage from neuronal insults. Phosphoglycerate mutase 5 (PGAM5) is localized to the mitochondrial outer-inner membrane contact sites, and the PGAM5-Drp1 pathway is involved in mitochondrial dysfunction and cell death. The purpose of this project was to evaluate the effects of PGAM5 on neuronal injury and mitochondrial dysfunction. PGAM5 was overexpressed in mice subjected to TBI and in primary cortical neurons injured by mechanical equiaxial stretching. PGAM5 deficiency alleviated neuroinflammation, blocked Parkin, PINK1, and Drp1 translocation to mitochondria and abnormal phosphorylation of Drp1, mitochondrial ultrastructural changes, and nerve malfunction in TBI mouse model. PGAM5-shRNA (short hairpin RNA) reduced Drp1 translocation and activation, including dephosphorylation of p-Drp1 on Ser622 (human Drp1 Ser616) and phosphorylation of Drp1 on Ser643 (human Drp1 Ser637). The levels of inflammatory cytokines, the degree of mitochondrial impairment (mitochondrial membrane potential, ADP/ATP, AMP/ADP, antioxidant capacity), and neuronal injury in stretch-induced primary cortical neurons were reduced by blocking expression of PGAM5. The inhibition of PGAM5 is neuroprotective attenuation of Drp1 activation, similar to that achieved by mitochondrial division inhibitor-1 (Mdivi1)-mediated Drp1 inhibition. Our findings demonstrate the critical role of PGAM5 in progression of neuronal injury from TBI Drp1 activation (dephosphorylation of p-Drp1 on Ser622 and phosphorylation of Drp1 on Ser643)-mediated mitochondrial dysfunction. The data may open a window for developing new drugs to prevent the neuropathology of TBI.