Extension of neurites requires the SNARE-dependent fusion of plasmalemmal precursor vesicles with the plasma membrane of growth cones. Here, we show that tomosyn localizes at the palm of growth cones and inhibits the fusion of the vesicles there, thus promoting transport of the vesicles to the plasma membrane of the leading edges of growth cones. Tomosyn localizes because ROCK activated by Rho small G protein phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the SNARE complex. In retraction of neurites, tomosyn localizes all over the edges of the neurites and inhibits fusion of the vesicles with the plasma membrane. Thus, tomosyn demarcates the plasma membrane by binding to syntaxin-1 phosphorylated by ROCK, and thereby regulates extension and retraction of neurites.