Neuropeptide secretion from dense-core vesicles (DCVs) controls many brain functions. Several components of the DCV exocytosis machinery have recently been identified, but the participation of a SEC1/MUNC18 (SM) protein has remained elusive. Here, we tested the ability of the three exocytic SM proteins expressed in the mammalian brain, MUNC18-1/2/3, to support neuropeptide secretion. We quantified DCV exocytosis at a single vesicle resolution on action potential (AP) train-stimulation in mouse CNS neurons (of unknown sex) using pHluorin-tagged and/or mCherry-tagged neuropeptide Y (NPY) or brain-derived neurotrophic factor (BDNF). Conditional inactivation of Munc18-1 abolished all DCV exocytosis. Expression of MUNC18-1, but not MUNC18-2 or MUNC18-3, supported DCV exocytosis in Munc18-1 null neurons. Heterozygous (HZ) inactivation of Munc18-1, as a model for reduced MUNC18-1 expression, impaired DCV exocytosis, especially during the initial phase of train-stimulation, when the release was maximal. These data show that neurons critically and selectively depend on MUNC18-1 for neuropeptide secretion. Impaired neuropeptide secretion may explain aspects of the behavioral and neurodevelopmental phenotypes that were observed in Munc18-1 HZ mice.