We currently lack a comprehensive understanding of the mechanisms underlying neural tube formation and their contributions to neural tube defects (NTDs). Developing a model to study such a complex morphogenetic process, especially one that models human-specific aspects, is critical. Three-dimensional, human embryonic stem cell (hESC)-derived neural rosettes (NRs) provide a powerful resource for in vitro modeling of human neural tube formation. Epigenomic maps reveal enhancer elements unique to NRs relative to 2D systems. A master regulatory network illustrates that key NR properties are related to their epigenomic landscapes. We found that folate-associated DNA methylation changes were enriched within NR regulatory elements near genes involved in neural tube formation and metabolism. Our comprehensive regulatory maps offer insights into the mechanisms by which folate may prevent NTDs. Lastly, our distal regulatory maps provide a better understanding of the potential role of neurological-disorder-associated SNPs. Display omitted •Neural rosettes have transcriptional and epigenomic landscapes distinct from 2D NSCs•Low methylated regions in NRs predict enhancers active later in neurodevelopment•NR regulatory maps provide insights into mechanisms of neural tube defects Neural rosettes offer a 3D model of neural tube development. Valensisi et al. show that these cells have distinct epigenomic landscapes compared to 2D neural stem cells. cis-regulatory elements identified in NRs harbor folate-associated CpGs, which may be important during neural tube development, and neurodevelopmental-disorder-associated variants.