Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.