Folate, choline and vitamin B12 are essential nutrients involved in one-carbon metabolism (OCM), a network of interconnected pathways necessary for the de novo synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Disruptions in OCM are associated with aberrant DNA synthesis and methylation and high risk for cancer. Thus, it is of particular importance to elucidate the role of these nutrients in the functioning of OCM. In addition, the status of these nutrients and their demand differ by reproductive and/or pathological state, further addressing the need to better understand the effects of these nutrients. This dissertation research involves both human participant studies and laboratory-based molecular research to advance current knowledge of the role of these nutrients. Study 1 examined the impact of mandatory folic acid fortification on DNA methylation status among postmenopausal women enrolled in the Women’s Health Initiative Observational Study (WHI-OS). As expected, given the role of folate in OCM, women with higher red blood cell (RBC) folate concentration had higher DNA methylation in the pre-fortification period. However, this expected result was not observed in the post-fortification period during which women with higher (vs. lower) RBC folate status had lower DNA methylation. Overall, these findings suggest an inverted U-shaped relationship between folate status and DNA methylation across fortification periods, and further investigation is warranted to clarify the health outcomes of the inverse relationship observed in the era of folic acid fortification. Study 2 examined associations between biomarkers of choline metabolism and colorectal cancer risk in a case-control study nested within the WHI-OS. The main findings indicate 1) a positive association between plasma trimethylamine N-oxide (TMAO; a derivative of choline produced by intestinal bacteria) and rectal cancer risk; and 2) an inverse relationship between plasma betaine and colorectal cancer risk. These findings demonstrate that alterations in choline metabolism associate with higher risk of colorectal cancer, suggesting the potential utilization of these biomarkers as predictors of increased colorectal cancer risk. Study 3 assessed changes in status and functional biomarkers of vitamin B12 among pregnant, lactating and control (nonpregnant, nonlactating) women who consumed equivalent vitamin B12 intakes under controlled feeding conditions. Pregnant (vs. control) women had a higher ratio of plasma holotranscobalamin (bioactive form of vitamin B12) to total vitamin B12, indicating that greater amounts of vitamin B12 are partitioned toward the biologically active form in this reproductive state. Overall, the results of this study suggest that metabolic adaptations transpire to enhance vitamin B12 supply during pregnancy. Study 4 employed in vitro cell culture models to investigate the effect of folate-independent generation of formate, a primary source of one-carbons for folate-mediated OCM, on the synthesis of purines and thymidylate. The study findings demonstrate that in human hepatocarcinoma (HepG2) cells, alcohol dehydrogenase 5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Taken together, this dissertation research spans from in vitro molecular studies to epidemiological studies to address the role of folate, choline and vitamin B12. The findings of this research will help inform the development of nutrient intake recommendations and the use of nutritional biomarkers for disease prediction.