Nitric oxide (NO) signalling contributes to many biological processes involved in activity-dependent fine tuning of neuronal communication. NO is involved in early developmental signalling of the nervous system and is associated with pathological pathways and age-related decline in neuronal function, thus playing a critical role in regulating neuronal function in physiology and disease. Here we assessed the effects of modulating endogenous neuronal nitric oxide synthase (NOS) activity on synaptic function, specifically on neurotransmitter release at the glutamatergic Drosophila neuromuscular junction (NMJ). We found that the absence of NOS activity enhanced synaptic release at the NMJ and conversely, overexpression of NOS diminished transmitter release. The effects of alterations in NO signalling are the consequence of acute signalling at the synapse as we did not observe any developmental changes in NMJ morphology or synaptic parameters, such as expression of the active zone protein, bruchpilot, which could account for changes in release. Ultrastructural analysis did not show any developmental effects in boutons from larvae with reduced NOS activity. Together, our data present evidence for a negative regulation of transmitter release by NO which has implications for physiological synaptic function but also pathological and age-related dysregulation of synaptic signalling.