AbstractAxon guidance in vertebrates is controlled by genetic cascades as well as by intrinsic activity-dependent refinement of connections. Midline axon crossing is one of the best studied pathfinding models and is fundamental to the establishment of bilaterally symmetric nervous systems. However, it is not known whether crossing requires intrinsic activity in axons, and what controls that activity. Further, a mechanism linking neuronal activity and gene expression has not been identified for axon pathfinding. Using embryonic zebrafish, we found that the NMDA receptor (NMDAR) NR1.1 subunit (grin1a) is expressed in commissural axons. Pharmacological inhibition ofgrin1a, hypoxia exposure reduction ofgrin1aexpression, or CRISPR knock-down ofgrin1aleads to defects in midline crossing. Inhibition of neuronal activity phenocopies the effects ofgrin1aloss on midline crossing. By combining pharmacological inhibition of the NMDAR with optogenetic stimulation to precisely restore neuronal activity, we observed rescue of midline crossing. This suggests that the NMDAR controls pathfinding by an activity-dependent mechanism. We further show that the NMDAR may act, via modulating activity, on the transcription factorarxa(mammalianArx), a known regulator of midline pathfinding. These findings uncover a novel role for the NMDAR in controlling activity to regulate commissural pathfinding and identifyarxaas a key link between the genetic and activity-dependent regulation of midline axon guidance.