Asymmetric elevation of the Ca2+ concentration in the growth cone can mediate both attractive and repulsive axon guidance. Ca2+ signals that are accompanied by Ca2+-induced Ca2+ release (CICR) trigger attraction, whereas Ca2+ signals that are not accompanied by CICR trigger repulsion. The molecular machinery downstream of Ca2+ signals, however, remains largely unknown. Here we report that asymmetric membrane trafficking mediates growth cone attraction. Local photolysis of caged Ca2+, together with CICR, on one side of the growth cone of a chick dorsal root ganglion neuron facilitated the microtubule-dependent centrifugal transport of vesicles towards the leading edge and their subsequent vesicle-associated membrane-protein 2 (VAMP2)-mediated exocytosis on the side with an elevated Ca2+ concentration. In contrast, Ca2+ signals without CICR had no effect on the vesicle transport. Furthermore, pharmacological inhibition of VAMP2-mediated exocytosis prevented growth cone attraction, but not repulsion. These results strongly suggest that growth cone attraction and repulsion are driven by distinct mechanisms, rather than using the same molecular machinery with opposing polarities.