Folic acid supplementation has been shown to significantly reduce both the occurrence and the recurrence of neural tube defects (NTDs) in human populations, yet the underlying mechanisms for reducing the risk of NTDs continue to be debated. This study examined genetic background and select folate metabolites as possible modifiers that may influence NTD risk. Specifically, several folate cycle and methylation metabolites were examined for their ability to reduce the occurrence of NTDs in two congenic mouse strains carrying targeted disruption of the folate receptor 1 (Folr1) gene. SWV-Folr1$^{tm1Fnn}$ and LM/Bc-Folr1$^{tm1Fnn}$ mice were provided with folate or several folate pathway metabolites, or combinations thereof, to determine the ability of these compounds to rescue nullizygous embryos from lethality and NTDs. Results demonstrated that SWV-Folr1$^{tm1Fnn}$ and LM/Bc-Folr1$^{tm1Fnn}$ mice exhibit different dose responses to folinic acid (5-formyl-tetrahydrofolate) supplementation; however, treating dams throughout gestation with downstream metabolites indicated that only folates rescued Folr1 nullizygous embryos from lethality and NTDs. Chemical inhibitors of folate metabolism were used to further elucidate essential enzymatic and biochemical metabolites. These data demonstrate that the inhibition of S-adenosyl-L-homocysteine hydrolase (AHCY) or selective inhibition of folate responsive isoprenylcysteine carboxylmethyltransferase (ICMT) results in embryo toxicity and fetuses with anterior NTDs. These data indicate that genetic background modifies NTD penetrance in folate-supplemented Folr1$^{tm1Fnn}$ mutants, while downstream metabolites of folate metabolism are not capable of rescuing Folr1$^{tm1Fnn}$ mutants. Moreover, these findings support the hypothesis that the methylation cycle and post-translational methylation of key signaling proteins such as Ras, by ICMT are essential to neural tube closure.