The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.