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Regulated secretion typically depends on activity-induced Ca2+ influx. However, in invertebrates, the endoplasmic reticulum (ER) plays a distinct role, particularly in the release of neuromodulators from dense-core vesicles (DCVs). Here, we investigated the role of the neuronal ER as a Ca2+ source for neuromodulator secretion in primary mouse neurons by directly monitoring ER and cytosolic Ca2+ dynamics, along with DCV exocytosis at single vesicle resolution. During neuronal activity, neurons with a low initial [Ca2+]ER took up Ca2+ into the ER, while those with a high initial [Ca2+]ER released ER Ca2+. These latter neurons showed more DCV exocytosis. Acute ER Ca2+ release by caffeine or thapsigargin application, resulted in minute increases in bulk cytosolic free Ca2+ that did not trigger DCV exocytosis. Remarkably, following ER Ca2+ depletion levels, activity-dependent Ca2+ influx and DCV exocytosis were reduced by 50-90%, while synaptic vesicle (SV) exocytosis was unaffected. L-type Ca2+-channel inhibition by nimodipine reduced DCV exocytosis and Ca2+ influx by 80-90 % without affecting SV exocytosis, a phenocopy of ER store depletion. In addition, introducing L-type channels lacking STIM1 interaction sites restored DCV fusion following ER store depletion. We conclude that the ER functions as a dynamic Ca2+ store serving both as a Ca2+ source or sink. Moreover, ER depletion activates a feedback loop that controls L-type Ca2+ channel activity, essential for DCV exocytosis.