Alcohol exposure during the formation and closure of the neural tube, or neurulation (embryonic day [E] 8-10 in mice; ∼4th week of human pregnancy), perturbs development of midline brain structures and significantly disrupts gene expression in the rostroventral neural tube (RVNT). Previously, alcohol exposure during neurulation was found to alter gene pathways related to cell proliferation, p53 signaling, ribosome biogenesis, immune signaling, organogenesis, and cell migration 6 or 24 h after administration. Our current study expands upon this work by investigating short-term gene expression changes in the RVNT following a single binge-like alcohol exposure during neurulation. Female C57BL/6J mice were administered a single dose of 2.9 g/kg alcohol or vehicle on E9.0 to target mid-neurulation. The RVNTs of stage-matched embryos were collected 2 or 4 h after exposure and processed for RNA-seq. Functional profiling was performed with g:Profiler, as well as with the CiliaCarta and DisGeNet databases. Two hours following E9.0 alcohol exposure, 650 genes in the RVNT were differentially expressed. Functional enrichment analysis revealed that pathways related to cellular metabolism, gene expression, cell cycle, organogenesis, and Hedgehog signaling were down-regulated, and pathways related to cellular stress response, p53 signaling, and hypoxia were up-regulated by alcohol. Four hours after alcohol exposure, 225 genes were differentially expressed. Biological processes related to metabolism, RNA binding, ribosome biogenesis, and methylation were down-regulated, while protein localization and binding, autophagy, and intracellular signaling pathways were up-regulated. Two hours after alcohol exposure, the differentially expressed genes were associated with disease terms related to eye and craniofacial development and anoxia. These data provide further information regarding the biological functions targeted by alcohol exposure during neurulation in regions of the neural tube that give rise to alcohol-sensitive midline brain structures. Disruption of these gene pathways contributes to the craniofacial and brain malformations associated with prenatal alcohol exposure.