Methylocella silvestris, an alphaproteobacterium isolated from a forest soil, can grow on trimethylamine N-oxide (TMAO) as a sole nitrogen source, however, the molecular and biochemical mechanisms underpinning its growth remain unknown. Marker-exchange mutagenesisenabled the identification of several genes involved in TMAO metabolism, including Msil_3606, a permease of the amino acids-polyamine (APC) superfamily, and Msil_3603, consisting of anN-terminal domain of unknown function (DUF1989) and a C-terminal tetrahydrofolate-binding domain. Null mutants of Msil_3603 and Msil_3606 can no longer grow on TMAO. Purified Msil_3603 from recombinant Escherichia coli can convert TMAO to dimethylamine and formaldehyde (1 TMAO 1 dimethylamine + 1 formaldehyde), confirming that it encodes a bona fide TMAO demethylase (Tdm). Tdm of M. silvestrisand eukaryotic TMAO demethylases have no sequence homology and contrasting characteristics. Recombinant Tdm of M. silvestris appears to be hexameric, has a high affinity for TMAO (Km= 3.3 mM; Vmax=21.7 nmolmin-1mg-1) and only catalyses demethylation of TMAO and a structural homologue, dimethyldodecylamine N-oxide. Our study has contributed to the understanding of the genetic and biochemical mechanisms for TMAO degradation in M. silvestris.