While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase (chata), vesicular acetylcholine transporters (vachta, vachtb), high-affinity choline transporter (hacta) and acetylcholinesterase (ache), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which continued during development. On the contrary, nAChR subunits remained highly expressed throughout the spinal cord. We found that protein and enzymatic activities of presynaptic cholinergic genes were also reduced in the rostral spinal cord. Our work demonstrating that cholinergic genes are initially expressed in the embryonic spinal cord, which is dynamically downregulated during development suggests that cholinergic signaling may play a pivotal role during the formation of intra-spinal locomotor circuit.