Estrogens comprise a group of related hormones occurring in predominantly female vertebrates, with endocrine disrupting and carcinogenic potential. Microbial biodegradation of estrogens is essential for their elimination from surface waters and wastewater. Aerobic bacteria employ oxygenases for the initial cleavage of the aromatic ring A. In contrast, anaerobic degradation of estrogens is initiated by methyl transfer-dependent conversion into androgens involving a putative cobalamin-dependent methyltransferase system. The methyl donor for this unprecedented reaction and its stoichiometric regeneration have remained unknown. With the biomass obtained from large-scale fermentation of an estrogen-degrading denitrifying bacterium, we identified S -adenosyl-methionine (SAM) as the methyl donor for the cobalamin-mediated methyl transfer to estrogens. To continuously supply C1 units to initiate estrogen degradation, genes for SAM regeneration from estradiol-derived catabolites are highly upregulated. Data presented here shed light into biochemical processes involved in the globally important microbial degradation of estrogens.