In the olfactory system of the sphinx moth Manduca sexta, migration of neuropil glial cells is triggered by olfactory receptor axons and depends on intraglial Ca2+ signaling. It is not known, however, how receptor axons and glial cells communicate and whether Ca2+ signaling is a consequence of this communication. We studied Ca2+ increases in glial cells in vivo and in situ, evoked by electrical stimulation of olfactory receptor axons in pupae and by odor stimulation of receptor neurons in adult moths. Axonal activity leads to Ca2+ increases in neuropil glial cells that are blocked by nicotinic acetylcholine receptor antagonists and can be mimicked by acetylcholine and carbachol application. In addition, Ca2+ transients were abolished by removal of external Ca2+ and blockage of voltage-gated Ca2+ channels. During development, acetylcholine-mediated Ca2+ signaling could first be elicited at stage 6, the time when neuropil glial cells start to migrate. Glial migration was reduced after injection of nicotinic antagonists into pupae. The results show that Ca2+ signaling can be induced by acetylcholine release from olfactory receptor axons, which activates nicotinic acetylcholine receptors and leads to voltage-gated Ca2+ influx. The results further suggest that cholinergic signaling in the olfactory system is required for glial cell migration in Manduca.