Selenoproteins contain the essential trace element selenium, the deficiency of which is associated with cancer or accelerated aging. Although selenoproteins are thought to be instrumental for the effects of selenium, the biological function of many of these proteins remains unknown. Here, we studied the role of selenoprotein T (SelT), a selenocysteine (Sec) -containing protein with no known function, which we have identified as a novel target gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) during PC12 cell differentiation. SelT was found to be ubiquitously expressed throughout embryonic development and in adulthood in rat. Immunocytochemical analysis revealed that SelT is mainly localized to the endoplasmic reticulum through a hydrophobic domain. PACAP and cAMP induced a rapid and long-lasting increase in SelT gene expression in PC12 cells, in a Ca²⁺-dependent manner. These results suggested a possible role of SelT in PACAP signaling during PC12 cell differentiation. Indeed, overexpression of SelT in PC12 cells provoked an increase in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) that was dependent on the Sec residue. Conversely, SelT gene knockdown inhibited the PACAP-induced increase in [Ca²⁺]_i and reduced hormone secretion. These findings demonstrate the implication of a selenoprotein in the regulation of Ca²⁺ homeostasis and neuroendocrine secretion in response to a cAMP-stimulating trophic factor.—Grumolato, L., Ghzili, H., Montero-Hadjadje, M., Gasman, S., Lesage, J., Tanguy, Y., Galas, L., Ait-Ali, D., Leprince, J., Guérineau, N. C., Elkahloun, A. G., Fournier, A., Vieau, D., Vaudry, H., Anouar, Y. Selenoprotein T is a PACAP-regulated gene involved in intracellular Ca²⁺ mobilization and neuroendocrine secretion.