This study investigates the role of the histone N-terminal acetyltransferase Naa40 in neural tube formation in mouse embryonic stem cells. Naa40 regulates gene expression and influences cell development through N-terminal acetylation of histones H2A and H4. Previous research has primarily focused on its involvement in cancer and metabolic disorders; however, its impact on nervous system development remains unclear. This study aims to clarify whether Naa40 affects neural tube formation through histone N-terminal acetylation, thereby providing further insights into its role in central nervous system development.We used Western blotting to confirm Naa40 expression in mouse embryonic stem cells (wild-type and Naa40 knockout) and to examine its effect on histone H4 acetylation. Using 3D cell culture techniques, we observed the process of neural tube formation and compared developmental differences between wild-type and Naa40 knockout cells. In addition, lentiviral infection was employed to reintroduce wild-type and mutant Naa40 for mechanism validation. Immunofluorescence staining was used to analyze cytoskeletal distribution and assess neural tube integrity.Our results show that Naa40 deletion abolishes histone H4 acetylation and disrupts normal neural tube development. Moreover, the Naa40 E139A mutation plays a particularly critical role in regulating neural development. This study aims to provide new insights into the epigenetic mechanisms underlying neural tube defects (NTDs) and serve as a reference for future research and therapeutic strategies related to these conditions.