Neurotransmitter release is generally considered to occur at active zones of synapses, and only in few instances, ectopic release of neurotransmitters has been demonstrated. However, the mechanism of ectopic release is poorly understood. We took advantage of the intimate morphological and functional proximity of olfactory receptor axons and specialized glial cells, olfactory ensheathing cells (OECs), to study ectopic neurotransmitter release. Stimulation of axons evoked purinergic and glutamatergic Ca²⁺ responses in OECs, indicating release of ATP and glutamate. In receptor axons expressing synapto-pHluorin, stimulation evoked an increase in synapto-pHluorin fluorescence, indicative for vesicle fusion. Transmitter release was dependent on Ca²⁺ and could be inhibited by suppressing vesicular neurotransmitter release with bafilomycin A1 and botulinum toxin A. Ca²⁺ transients in OECs evoked by ATP and laser photolysis of NP-EGTA resulted in constriction of adjacent blood vessels. Our results indicate that ATP and glutamate are ectopically released from vesicles along axons and mediate neurovascular coupling via glial Ca²⁺ signaling.