Mammalian serine hydroxymethyltransferase (SHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate in the cytoplasm (cSHMT, SHMT1) and mitochondria (mSHMT, SHMT2). cSHMT preferentially supplies one-carbon units for thymidylate biosynthesis, depletes methylenetetrahydrofolate pools for S-adenosylmethionine (SAM) synthesis by synthesizing serine, sequesters 5-methyltetrahydrofolate, and inhibits SAM synthesis.