Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.