Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.