Traumatic brain injury (TBI) is the leading cause of death and disability worldwide. Calpains, a family of cysteine proteases have been implicated in cells death following TBI. Using immunohistochemistry calpain expression was analyzed in post mortem brain tissue obtained from patients who died after TBI, and findings were compared with the brain tissue from patients who died from sudden cardiac arrest. In the injured cortex an increase in calpain expression was observed in all resident brain cells: neurons, glial and endothelial cells in comparison to the control group (all p < 0.001). Calpain expression was analyzed in different post-traumatic intervals, from day 0 until 10 days post-injury, in order to establish a time course of expression in the brain cortex after TBI. Expression was detected in the cortex 5 hours after the accident, peaked at 72 hours, and substantially reduced by 10 days after TBI. Calpain expression in the cortex significantly changed during the time from TBI to death (p < 0.001), and the most prominent expression was detected in the cortex 3 days after TBI. Our results indicate that prolonged calpain expression in resident brain cells (neurons, glial and endothelial cells) plays an important role in neuronal degeneration following TBI.