Calpains are cysteine proteases activated in response to intracellular calcium signaling. Activated calpains regulate various cellular functions by degrading substrate molecules in a site-specific manner. Although most calpains are localized in the cytosol, we previously reported that calpain-5 exists in the mitochondria. The mitochondrial calpain-5 is activated during endoplasmic reticulum (ER) stress. However, the substrate of calpain-5, as well as the physiological significance of calpain-5 activation, has not yet been elucidated. In the present study, we treated HeLa cells with A23187, tunicamycin, or hydrogen peroxide to induce intracellular calcium increase, resulting in cell death. The cells treated with A23187 or tunicamycin exhibited the activation of calpain-5 and truncation of caspase-4. The truncation of caspase-4 was inhibited by the repression of calpain-5 expression with the appropriate siRNA. Additionally, both calpain-5 and caspase-4 were observed in the mitochondria. Our study is the first to demonstrate that the activation of mitochondrial calpain-5 triggers the truncation of caspase-4, suggesting that mitochondrial calpain-5 regulates the downstream pathway of caspase-4, including cell death and the inflammatory cascade. The results of the present study provide new insights into ER-stress-related diseases such as Alzheimer's disease and cancer. These perspectives allow us to propose new therapeutic strategies such as the development of inhibitors or activators of calpain-5, which may be useful in the development of treatment for ER-stress-related diseases.