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Summary In the surface waters of the warm oligotrophic ocean, filaments and aggregated colonies of the nitrogen (N)‐fixing cyanobacteriumTrichodesmiumcreate microscale nutrient‐rich oases. These hotspots fuel primary productivity and harbour a diverse consortium of heterotrophs. Interactions with associated microbiota can affect the physiology ofTrichodesmium, often in ways that have been predicted to support its growth. Recently, it was found that trimethylamine (TMA), a globally abundant organic N compound, inhibits N₂fixation in cultures ofTrichodesmiumwithout impairing growth rate, suggesting thatTrichodesmiumcan use TMA as an alternate N source. In this study,¹⁵N‐TMA DNA stable isotope probing (SIP) of aTrichodesmiumenrichment was employed to further investigate TMA metabolism and determine whether TMA‐N is incorporated directly or secondarily via cross‐feeding facilitated by microbial associates. Herein, we identify two members of the marineRoseobacterclade (MRC) of Alphaproteobacteria as the likely metabolizers of TMA and provide genomic evidence that they converted TMA into a more readily available form of N, e.g., ammonium (NH₄⁺), which was subsequently used byTrichodesmiumand the rest of the community. The results implicate microbiome‐mediated carbon (C) and N transformations in modulating N₂fixation and thus highlight the involvement of host‐associated heterotrophs in global biogeochemical cycling.