5,10-Methylenetetrahydrofolate plays a critical role in one-carbon metabolism, serving as a key intermediate in the synthesis of nucleotides. It is essential for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a process crucial for DNA replication and repair. This makes it vital for cell division and growth, particularly in rapidly dividing cells like those in bone marrow and the intestinal lining.

In medical research, it is studied for its role in folate metabolism and its potential impact on conditions like cancer, cardiovascular diseases, and neural tube defects. It is also relevant in understanding the efficacy of antifolate drugs, which target folate-dependent pathways in cancer and bacterial infections. Additionally, its levels are monitored in cases of folate deficiency or genetic mutations affecting folate metabolism, such as MTHFR gene variants.