Trimethylamine N-oxide (TMAO) is a common osmolyte found in a variety of marine biota and has been detected at nanomolar concentrations in oceanic surface waters. TMAO can serve as an important nutrient for ecologically important marine heterotrophic bacteria, particularly the SAR11 clade and marine Roseobacter clade (MRC). However, the enzymes responsible for TMAO catabolism and the membrane transporter required for TMAO uptake into microbial cells have yet to be identified. We show here that the enzyme TMAO demethylase (Tdm) catalyzes the first step in TMAO degradation. This enzyme represents a large group of proteins with an uncharacterized domain (DUF1989). The function of TMAO demethylase in a representative from the SAR11 clade (strain HIMB59) and in a representative of the MRC (Ruegeria pomeroyi DSS-3) was confirmed by heterologous expression of tdm (the gene encoding Tdm) in Escherichia coli. In R. pomeroyi, mutagenesis experiments confirmed that tdm is essential for growth on TMAO. We also identified a unique ATP-binding cassette transporter (TmoXWV) found in a variety of marine bacteria and experimentally confirmed its specificity for TMAO through marker exchange mutagenesis and lacZ reporter assays of the promoter for genes encoding this transporter. Both Tdm and TmoXWV are particularly abundant in natural seawater assemblages and actively expressed, as indicated by a number of recent metatranscriptomic and metaproteomic studies. These data suggest that TMAO represents a significant, yet overlooked, nutrient for marine bacteria.