Many studies have focused on Ca²⁺ as the instructive, polarizing signal for growth cone turning. Conversely, the cyclic nucleotides, cAMP and cGMP, have been thought to play a permissive role by uniformly regulating the activity of Ca²⁺ channels throughout the growth cone. However, asymmetric cAMP or cGMP elevations are sufficient for inducing growth cone turning, as indicative of their instructive roles in axon guidance. Here, we show that increases in cAMP and cGMP antagonistically control microtubule (MT)-dependent vesicle transport, which in turn causes growth cone turning. Embryonic chick dorsal root ganglion neuron growth cones showed attraction or repulsion, respectively, when cAMP or cGMP concentration was locally increased on one side of the growth cone by photolysis of caged compounds. These turning responses were blocked by pharmacological and genetical perturbations of MT dynamics and of the function of vesicle associated membrane protein (VAMP)7. Time-lapse imaging of end binding protein 1 revealed that cAMP elevation on one side of the growth cone ipsilaterally increased the frequency of MT contact with the leading edge. The opposite was true for cGMP: MT contact ipsilaterally decreased with cGMP elevation. These effects on MT dynamics in turn led to spatial asymmetry in the frequency of VAMP7-positive vesicle transport toward the leading edge, i.e. cAMP ipsilaterally increased, while cGMP decreased, transport. Next we examined whether these signaling machineries were involved in physiological guidance cue-induced turning. An extracellular gradient of pituitary adenylate cyclase-activating polypeptide (PACAP) caused asymmetric cAMP elevation in the growth cone; higher increases on the side facing the source of PACAP. This cAMP elevation ipsilaterally increased the frequency of MT contact with the leading edge and VAMP7-vesicle transport. Furthermore, PACAP failed to attract the growth cones expressing the inhibitory domain of VAMP7. Our finding that antagonistic control of MT and membrane dynamics by cAMP and cGMP provides new insights into the mechanisms of guided migration and of polarity formation.