Secretion principles are conserved from yeast to humans, and many yeast orthologs have established roles in synaptic vesicle exocytosis in the mammalian brain. Surprisingly, SEC4 orthologs and their effectors, the exocyst, are dispensable for synaptic vesicle exocytosis. Here, we identify the SEC4 ortholog RAB3 and its neuronal effector, RIM1, as essential molecules for neuropeptide and neurotrophin release from dense-core vesicles (DCVs) in mammalian neurons. Inactivation of all four RAB3 genes nearly ablated DCV exocytosis, and re-expression of RAB3A restored this deficit. In RIM1/2-deficient neurons, DCV exocytosis was undetectable. Full-length RIM1, but not mutants that lack RAB3 or MUNC13 binding, restored release. Strikingly, a short N-terminal RIM1 fragment only harboring RAB3- and MUNC13-interacting domains was sufficient to support DCV exocytosis. We propose that RIM and MUNC13 emerged as mammalian alternatives to the yeast exocyst complex as essential RAB3/SEC4 effectors and organizers of DCV fusion sites by recruiting DCVs via RAB3.