Neural tube closure defects (NTDs) are common developmental anomalies that result from failure of the embryonic neural tube to bend and fuse during development, resulting in herniation and deterioration of nervous system tissue. Low folate status is an important environmental determinant of NTD risk, and maternal folate supplementation can prevent the occurrence of NTDs by 50-70%, but the mechanisms remain unknown. Folates function to carry and activate methyl groups for a set of anabolic reactions collectively known as one-carbon metabolism (OCM). OCM is required for the de novo biosynthesis of purines and thymylate, and for the remethylation of homocysteine to methionine. Methionine can be adenosylated to form S-adenosylmethionine, which is the universal methyl donor for cellular methylation reactions, including chromatin methylation. Impairments in OCM result in elevated homocysteine, reduced proliferation, genomic instability, and chromatin hypomethylation. Evidence from human genetic studies indicates that NTDs are complex traits that arise from deleterious gene-nutrient interactions, although human gene candidates that account for population-wide risk have not yet been identified. In these studies, we explore NTDs in response to disruption of two folate-dependent genes, cytoplasmic serine hydroxymethyltransferase (Shmt1) and the gene encoding the trifunctional enzyme MTHFD1 (Mthfd1). SHMT1 regulates the partitioning of one-carbons by prioritizing thymidylate biosynthesis at the expense of cellular methylation. MTHFD1 catalyzes the synthesis of 10-formylTHF, which is required for de novo purine biosynthesis. Disruption of Shmt1 resulted in NTDs in mice that mimicked folateresponsive NTDs in humans. This is first gene within folate metabolism that when disrupted causes NTDs in mice. Shmt1 disruption in the splotch mutant, a folateresponsive NTD model with impaired thymidylate biosynthesis, exacerbated and increased the frequency of NTDs, further implicating impaired thymidylate biosynthesis in NTD pathogenesis. Similarly, disruption of MTHFD1 resulted in developmental anomalies associated with the maternal, not fetal, genotype, as observed in humans. Supplementation of Mthfd1-deficient dams with hypoxanthine also revealed NTDs in Mthfd1-deficient embryos. These data implicate impaired nucleotide biosynthesis in NTD pathogenesis and highlight two new mouse models to study mechanisms underlying folate-responsive developmental anomalies.