Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures ( = 5,  < 0.05). The PrP-hCAST mice ( = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls ( = 9,  < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume ( < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls ( < 0.05,  = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.