The formation of axon tracts depends on the spatial accuracy of growth cone navigation. Extracellular guidance cues attract or repel an axon via asymmetric elevation of cytosolic Ca²⁺ concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca²⁺ elevation, respectively. The distinction between attractive and repulsive Ca²⁺ signals resides in their source: Ca²⁺ release from the endoplasmic reticulum (ER) for attraction and Ca²⁺ influx from the extracellular space for repulsion. Downstream of these Ca²⁺ signals, local activation of membrane trafficking steers the growth cone bidirectionally, with exocytosis driving attraction and endocytosis causing repulsion. However, it remains unknown how growth cones can discriminate these Ca²⁺ signals to operate opposite trafficking events. Here we have conducted a large-scale search for Ca²⁺ effectors that associate with two types of ER Ca²⁺ channels, ryanodine receptors (RyR) or IP₃ receptors (IP₃R), and identified myosin Va (MyoVa) as a critical effector for attractive guidance. We have accumulated evidence that MyoVa can respond specifically to ER-derived Ca²⁺ and trigger exocytic vesicle transport for attraction. First, MyoVa dissociates from RyR in response to high Ca²⁺, suggesting that MyoVa can detect Ca²⁺ microdomains on the ER. Second, MyoVa binding to ER Ca²⁺ channels is a prerequisite for Ca²⁺-triggered exocytic vesicle transport and growth cone attraction. Third, forced dissociation of MyoVa from ER Ca²⁺ channels using photoactivatable peptides can mimic attractive Ca²⁺ signals and elicit exocytosis. Importantly, this manipulation on one side of the growth cone is sufficient to initiate attractive turning. Finally, MyoVa binding to ER Ca²⁺ channels participates in axon guidance in vivo. Thus, we have discovered a novel mechanism for sensing ER-derived Ca²⁺, highlighting the functional significance of spatial confinement of Ca²⁺ signals in controlling the polarity of cell migration.