Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross-talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin-1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin-1 (STX-1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss-of-function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock-down of Syntaxin-1, VAMP-2, and SNAP-25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol, 2017.