Folate (vitamin B9) and vitamin B12 are essential nutrients that work together to support DNA replication and cell division. Recent concern has been raised over the potential for adverse health outcomes in individuals with excess folate exposure and low B12 status, but the molecular basis for these suggested links has not been evaluated. Further, the relationship between folate and B12 has been studied for decades- but primarily in the context of nuclear DNA synthesis and stability. This dissertation explores the interaction between folate and B12 in the context of excess folic acid and low B12, as well as the impact these nutrients have on mitochondrial DNA (mtDNA) stability. We observed that even when consumed at high doses, folic acid does not meaningfully accumulate in mouse tissues, though high-dose folic acid shifted folate vitamer distribution and elevated uracil in nuclear DNA of colon tissue. High-dose folic acid exposure did not affect liver total folate concentrations, vitamer distribution, or uracil levels in nuclear DNA. Additionally, we found that reduced expression of the B12-dependent enzyme methionine synthase (Mtr) caused uracil accumulation in mtDNA and impaired mitochondrial function in mouse liver. Further, we validated that dietary B12 deficiency led to uracil accumulation in mtDNA and impaired mitochondrial function in mouse skeletal muscle. We concluded that excess folic acid exposure has tissue-specific effects and that folate and B12 are essential to not only nuclear but also mtDNA integrity.