Synaptic vesicles (SVs) release neurotransmitters at specialized active zones, but release sites and organizing principles for the other major secretory pathway, neuropeptide/neuromodulator release from dense-core vesicles (DCVs), remain elusive. Here, we identify dynamins, yeast Vps1 orthologs, as important DCV fusion site organizers in mammalian CNS neurons. Genetic or pharmacological inactivation of all three dynamins strongly impaired DCV exocytosis, while SV exocytosis remained unaffected. Wildtype dynamin restored normal exocytosis, but not GTPase-deficient or membrane-binding mutants that cause neurodevelopmental syndromes. During prolonged stimulation, repeated use of the same DCV fusion location was impaired in dynamin 1-3 triple knockout neurons. The syntaxin-1 staining efficiency, but not its expression level, was reduced. αSNAP expression restored this. We conclude that mammalian dynamins organize DCV fusion sites, downstream of αSNAP-mediated cis-SNARE disassembly, by regulating the equilibrium between fusogenic and non-fusogenic syntaxin-1 and promoting its availability to form trans-SNARE complexes that drive DCV exocytosis.