Calpains are calcium (Ca2+) activated neutral proteases that are involved in several essential signaling pathways. One Calpain-specific proteolytic target is Junctophilin-2 (JP2), an important structural protein of Ca2+ release units required for the excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in mouse models of heart failure has been reported previously, the precise molecular identity of the Calpain cleavage sites and the (patho-) physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565 but subsequently also at three additional secondary Calpain cleavage sites. We identified the Calpain-specific primary cleavage products not only in mouse but also in human iPSC-derived cardiomyocytes (hiPSC-CMs). Knockout of RyR2 in hiPSC-CMs destabilized JP2 resulting in an increase of the Calpain-specifc cleavage fragments. The primary N-terminal cleavage product (NT1) accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+ dependent manner, closely associated with DNA-rich chromosomal regions, where NT1 is proposed to function as a cardioprotective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.