Nitric oxide (NO) is a signaling molecule that is synthesized in a range of tissues by the NO synthases (NOSs). In the immature nervous system, the neuronal isoform of NOS (NOS1) is often expressed during periods of axon outgrowth and elaboration. However, there is little direct molecular evidence to suggest that NOS1 influences these processes. Here we address the functional role of NOS1 during in vivo zebrafish locomotor circuit development. We show that NOS1 is expressed in a population of interneurons that lie close to nascent motoneurons of the spinal cord. To determine how this protein regulates spinal network assembly, we perturbed NOS1 expression in vivo with antisense morpholino oligonucleotides. This treatment dramatically increased the number of axon collaterals formed by motoneuron axons, an effect mimicked by pharmacological inhibition of the NO/cGMP signaling pathway. In contrast, exogenous elevation of NO/cGMP levels suppressed motor axon branching. These effects were not accompanied by a change in motoneuron number, suggesting that NOS1 does not regulate motoneuron differentiation. Finally we show that perturbation of NO signaling affects the ontogeny of locomotor performance. Our findings provide evidence that NOS1 is a key regulator of motor axon ontogeny in the developing vertebrate spinal cord.