ABSTRACT Congenital defects are the leading cause of neonatal mortality, resulting in more infant deaths than the combined adult death tolls of Alzheimer’s disease, strokes, diabetes, and influenza. Neural tube defects (NTDs), the second most common category of human birth defects, arise when the neural tube fails to close properly during neurulation. Globally, these defects are estimated to affect approximately 18.6 per 10,000 live births and the prevalence of NTDs is 1–2 per 1,000 births in most regions of the US. There are approximately 2,300 NTD- affected pregnancies in the US each year, whose lifetime medical costs are estimated to be $560,000 per child or $1.68 billion per year nationwide. Despite intensive investigation for decades, relatively little is known about the underlying NTD risk factors. It is generally accepted that NTDs are of a multi-factorial origin, having both environmental and genetic factors that contribute to the malformation. Although it is established that periconceptional use of folic acid (FA) prevents a significant percentage of the population burden of NTDs, the mechanisms underlying those processes by which FA reduces NTD risk remains unknown. Importantly, there are significant numbers of NTDs that are not preventable by FA supplementation, with these FA-resistant NTDs occurring at an apparent baseline rate of 5 per 10,000 live births. Thus, NTDs remain a substantial public health problem, and there is a critical need to understand the mechanisms underlying FA-resistant NTDs and to develop novel intervention strategies targeting this population To expand upon work performed in the initial funding period, our proposed line of study explores mechanisms by which impairment of mitochondrial one carbon metabolism causes NTDs and how our proposed interventions successfully restore proper NTC. Simultaneously, we are testing the efficacy and investigating mechanisms by which glycine supplementation rescues these FA-resistant defects. Establishing these mechanisms and relating them to actual human NTD variants may eventually allow us to utilize our proposed intervention strategies to prevent previously unpreventable birth defects by informing public health policy or precision medicine strategies, thus reducing the significant negative health burden of these debilitating defects on affected families and the public.