DHFR catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, a critical cofactor in one-carbon metabolism 1. This enzyme is essential for de novo synthesis of thymidylate, purines, and glycine, supporting DNA replication and cell division 2. DHFR functions in both nuclear and mitochondrial pathways, and uniquely binds its own mRNA to regulate translation 3. Clinically, DHFR deficiency causes megaloblastic anemia, reflecting impaired DNA synthesis in rapidly dividing cells. DHFR gene amplification is a major mechanism of methotrexate (MTX) resistance in cancer, evolving through homogenously staining regions and extrachromosomal DNA 4. Conversely, MTX-based PROTACs selectively degrade DHFR, offering potential therapeutic strategies with distinct phenotypes compared to conventional MTX inhibition 5. Beyond cancer, endothelial DHFR dysfunction contributes to pulmonary hypertension through impaired tetrahydrobiopterin metabolism and eNOS uncoupling; DHFR overexpression reversed pathophysiological features in mouse models 6. Additionally, DHFR polymorphisms associate with neural tube defect susceptibility through altered folate metabolism 7. DHFR's genomic location upstream of MSH3 creates a shared regulatory region amplified during chemotherapy, linking metabolic and DNA repair pathways with implications for mutation rates and drug sensitivity.