AbstractThe induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria‐localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia–reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.