Dihydrofolate reductase activity is required in One Carbon Metabolism to ensure that the biologically active form of folate, tetrahydrofolate, is replenished and available as an enzyme cofactor for numerous cellular reactions, including purine and pyrimidine synthesis. Most cellular enzyme activity was thought to arise from the product of the DHFR gene on chromosome 5, with its paralogue DHFR2 (formerly known as DHFRL1
[chromosome 3]), believed to be responsible for mitochondrial dihydrofolate activity based on recombinant versions of the enzyme. In this paper, we confirm our earlier findings that dihydrofolate reductase activity in mitochondria is derived from the DHFR gene rather than DHFR2 and that endogenous DHFR2 protein is not detectable in most cells and tissues. Using HepG2 cell lines with modulated expression of either DHFR or DHFR2, we observed an impact of DHFR2 RNA on One Carbon Metabolism mediated through an influence on DHFR expression and activity. Knockout of DHFR2 results in a drop in dihydrofolate reductase activity, lowered 10-formyltetrahydrofolate abundance, downregulation of DHFR mRNA, and diminished DHFR protein abundance. We also observed downregulation of Serine Hydroxymethyltransferase and Thymidylate Synthase, two One Carbon Metabolism enzymes that work with DHFR to support de novo thymidylate synthesis. The expression of recombinant DHFR2 resulted in restoration of DHFR mRNA and protein levels while a DHFR knockdown cell line showed upregulation of DHFR2 RNA. We propose that the DHFR2 gene encodes an RNA molecule that regulates cellular dihydrofolate reductase activity through its impact on DHFR mRNA and protein.