Calcium-dependent proteolytic calpains are implicated in a variety of physiological processes, as well as pathologies associated with calcium overload. However, the mechanism by which calpain is activated remains elusive since intracellular calcium levels under physiological conditions do not reach the high concentration range required to trigger calpain activation. From a candidate screen using the abundance of the calpain target glutamate receptor GluRIIA at the Drosophila neuromuscular junction as readout, we uncovered that calpain activity was inhibited upon knockdown of Ttm50, a subunit of the Tim23 complex involved in the import of proteins across the mitochondrial inner membrane. Unexpectedly, Ttm50 and calpain are co-located at calcium stores Golgi and endoplasmic reticulum (ER), and Ttm50 interacts with calpain via its C-terminus. This interaction is required for calpain localization at Golgi/ER, and increases calcium sensitivity of calpain by roughly an order of magnitude.