5,10-Methylenetetrahydrofolate (5,10-CH₂-THF) is a crucial intermediate in the one-carbon metabolism pathway of folate. It primarily functions as a methylene group donor in the synthesis of thymidylate (dTMP) from deoxyuridylate (dUMP), catalyzed by thymidylate synthase. This reaction is vital for DNA synthesis, repair, and cell proliferation, making it essential for growth and a target in anticancer therapies. Additionally, 5,10-CH₂-THF is converted to 5-methyltetrahydrofolate by the enzyme methylenetetrahydrofolate reductase (MTHFR), which then serves as the methyl donor in the remethylation of homocysteine to methionine. This process supports amino acid metabolism, protein synthesis, and is linked to cardiovascular and neurological health. Disruptions in this pathway, such as MTHFR polymorphisms, are associated with elevated homocysteine levels, neural tube defects, and increased risk of cancer and genetic disorders. It is widely used in biochemical research and nutritional supplementation for folate-related conditions.