Folates, in the form of tetrahydrolates, serve as cofactors for enzymes in a biological network of metabolic pathways that are necessary for the de novo synthesis of purines, thymidylate, and the vitamin B12 dependent-remethylation of homocysteine to methionine. Impaired folate-mediated one-carbon metabolism has been identified as a risk factor for neurodegeneration and disrupted nerve function, however the pathway(s) are not clearly defined. Serine hydroxymethyltransferase 1 (SHMT1) is a folate-dependent enzyme that serves as a scaffold protein for the assembly of a metabolic multi-enzyme complex that regulates de novo thymidylate synthesis. Disrupted Shmt1 expression sensitizes folate-deficient mice to develop folic-acid responsive neural tube defects. Therefore, studying the consequences of impairments in both Shmt1 expression and one-carbon metabolism may clarify the mechanisms by which folate and vitamin B12 deficiencies contribute to peripheral neuropathy. This research used a mouse model of decreased Shmt1 expression to identify underlying mechanisms linking impaired folate and vitamin B12 metabolism and neuropathy. First, a mouse model of disrupted Shmt1 expression was used to elucidate the role of impaired de novo thymidylate (dTMP) synthesis in vitamin B12 deficiency-induced neurodegeneration in the peripheral and central nervous system. Second, the mouse model of disrupted Shmt1 expression leading to disrupted de novo dTMP synthesis and a second diabetic model of diabetes was used to investigate the role of uracil misincorporation and DNA damage in peripheral neuropathy. Differences in motor coordination and sensory function in aged Shmt1+/+ and Shmt1+/- mice fed vitamin B12 and folic acid replete and deficient diets were measured using the accelerated rotarod, staggered rotarod, and tail flick test. Multiple linear and logistic regressive analyses indicated that Shmt1+/- mice fed vitamin B12 and folic acid deficient diets, as well as dietary folic acid and nucleoside supplemented diets, had significantly lower staggered rotarod motor performance and delayed latencies to tail flick compared to Shmt1+/- mice on a control diet. Additionally, dietary deoxyuridine and uridine supplementation did not rescue the impaired motor and sensory effects of vitamin B12 and folic acid deficiency in Shmt1+/- mice. Motor and sensory nerve conduction was measured in peripheral nerve tissue of Shmt1+/+, Shmt1+/-, db/db, and Shmt1+/-; db/db mice fed vitamin B12 and folic acid deficient and replete diets. Surprisingly, Shmt1 heterozygosity significantly impaired motor nerve conduction velocity in the sciatic nerve of mice fed both control and folic acid and vitamin B12 deficient diets. Together, these experiments were novel in their approach of using mouse models of impaired folate mediated one-carbon metabolism to identify a causal relationship between disrupted de novo dTMP synthesis and neuropathy. These findings implicate DNA damage and repair as potential targets of treatment for populations at risk of peripheral neuropathy.