Updated project metadata. One-carbon metabolism is a universal hub for cellular metabolism and epigenetic regulation.1–3 Here, we report that formaldehyde (FA), a one-carbon unit that organisms produce in substantial quantities through folate metabolism,4 is a regulator of the one-carbon cycle via the biosynthesis of S-adenosyl-L-methionine (SAM), an essential one-carbon building block for synthesis of nucleotides, amino acids, and methylated nucleic acids and proteins.5 Activity-based protein profiling (ABPP) in mouse liver tissue identifies FA-sensitive cysteine sites across the proteome, revealing several one-carbon cycle targets including S-adenosylmethionine synthetase isoform 1 (MAT1A), the terminal enzyme in SAM biosynthesis. Biochemical studies of the formaldehyde-MAT1A interaction establish FA-dependent inhibition of MAT1A activity through a conserved C120 site, as the MAT2A isoform lacking this cysteine is not FA-sensitive. CRISPR knockout-generated HepG2 cell models that predominantly express either MAT1A or MAT2A show that MAT1A-positive cells respond to FA treatment in a dose-dependent manner by decreasing their SAM levels and downstream RNA methylation, whereas the MAT2A-positive cells are not affected by FA. Our findings reveal an unexpected interplay between SAM and FA, two central one-carbon units to influence the overall methylation potential of the cell.