During the process of synapse formation, thousands of proteins assemble at prospective sites of cell-cell communication. Although many of these proteins have been identified, the roles they play in generating functional connections during development remain unknown. 4.1 scaffolding proteins have been implicated in synapse formation and maturation in vitro, but in vivo studies for some family members have suggested these proteins are not important for this role. We examined the role of family member 4.1B because it has been implicated in glutamatergic synaptogenesis, but has not been described in vivo. We identified two 4.1B genes in zebrafish, 4.1Ba and 4.1Bb, by sequence comparisons and synteny analysis. In situ hybridization shows these genes are differentially expressed, with 4.1Ba expressed primarily in the nervous system and 4.1Bb expressed in the nervous system and muscle, but not the spinal cord. We focused our studies on 4.1Ba in the spinal cord. 4.1Ba knockdown reduced the number of glutamatergic synapses at caudal primary motor neurons and caused an increase in the duration of touch-evoked coiling. These results suggest 4.1Ba is important for the formation of functional glutamatergic synapses in the developing zebrafish spinal cord.