Vitamin B12-associated pathologies are common, but the underlying mechanisms are unclear. In the cytosol, vitamin B12 functions as a required cofactor for methionine synthase (MTR) in the remethylation of homocysteine to methionine, which regenerates tetrahydrofolate (THF) for subsequent nucleotide biosyntheses. Impairments in this pathway are most apparent in megaloblastic anemia, which results from the inhibition of DNA synthesis due to the trapping of folate cofactors as 5-methyltetrahydrofolate (5-methylTHF). The first objective sought to determine the impact of maternal vitamin B12 deficiency and Shmt1 disruption on neural tube defect (NTD) incidence. Vitamin B12 deficiency, independently of folate status, caused NTDs in Shmt1-/- and Shmt1-/+ embryos. Folate status and folate-deficiency in vitamin B12 deficiency did not affect NTD incidence. Folate, but not vitamin B12 deficiency, significantly increased plasma homocysteine in pregnant dams and resulted in decreased embryonic growth. Taken together, these findings suggest that neural tube closure is more sensitive to maternal vitamin B12 deficiency than homocysteine remethylation or embryonic growth. The second objective aimed to determine the impact of a cytosolic vitamin B12 deficiency on nuclear de novo thymidylate (dTMP) biosynthesis, given the recent discovery of a nuclear, multi-protein complex for de novo synthesis of dTMP. Both human fibroblasts with loss-of-function mutations in MTR and HeLa cells treated with nitrous oxide (N2O) gas demonstrated intracellular accumulation of 5-methylTHF ranging from 1.75-2.5-fold compared to control conditions. In vitamin B12 depleted HeLa cells, the nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with a greater than 4-fold increase observed at the expense of THF. Vitamin B12 depletion in HeLa cells impaired rates of de novo dTMP biosynthesis and increased DNA damage; both outcomes were exacerbated by folate depletion. By contrast, MTR loss-of-function depressed rates of de novo dTMP biosynthesis, but did not increase DNA damage; this latter observation may be explained by the fact that specific tissues exhibit differential sensitivity to vitamin B12 deficiency. Taken together, these findings demonstrate cytosolic vitamin B12 depletion traps nuclear folate as 5-methylTHF, decreases rates of de novo dTMP biosynthesis, and increases DNA damage, providing a mechanism that can be extended to vitamin B12-associated pathologies.