BACKGROUND: Sialyllacto-N-tetraose b (LSTb) is a component of human milk oligosaccharides. Due to its low concentration, the impact of LSTb on neurodevelopment remains largely unexplored. It is worth studying whether LSTb should be added to infant formula to simulate breast milk. This study aimed to investigate the effect of LSTb on the development of motor neurons of the central nervous system using a transgenic zebrafish model. METHODS: Transgenic (Tg) zebrafish line (Hb9:GFP) was incubated with LSTb, and the axonal growth of caudal primary (CaP) neurons was assessed. Locomotor behavior was evaluated, and RNA sequencing (RNA-seq) was performed to identify the differentially expressed genes (DEGs). The expression of and , genes involved in axon guidance, was further analyzed through real-time polymerase chain reaction (real-time PCR) and whole-mount hybridization. RESULTS: There was a significant increase in the number and length of CaP axon branches, suggesting that LSTb promotes CaP development. Behavioral analysis revealed enhanced locomotor activity in LSTb-treated larvae, indicating improved motor function. RNA-seq analysis identified 5,847 DEGs related to central nervous system neuron differentiation, including and , which are known to contribute to axon guidance. In situ hybridization confirmed increased expression in the central nervous system of LSTb-treated larvae. CONCLUSIONS: LSTb significantly influences motor neuron development, potentially through the upregulation of and . This research provides valuable insights into the role of LSTb in neurodevelopment.