SummaryGlycine betaine (GBT) is a compatible solute in high concentrations in marine microorganisms. As a component of labile organic matter, GBT has complex biochemical potential as a substrate for microbial use that is unconstrained in the environment. Here we determine the uptake kinetics and metabolic fate of GBT in two natural microbial communities in the North Pacific characterized by different nitrate concentrations. Dissolved GBT had maximum uptake rates of 0.36 and 0.56 nM h−1 with half‐saturation constants of 79 and 11 nM in the high nitrate and low nitrate stations respectively. During multiday incubations, most GBT taken into cells was retained as a compatible solute. Stable isotopes derived from the added GBT were also observed in other metabolites, including choline, carnitine and sarcosine, suggesting that GBT was used for biosynthesis and for catabolism to pyruvate and ammonium. Where nitrate was scarce, GBT was primarily metabolized via demethylation to glycine. Gene transcript data were consistent with SAR11 using GBT as a source of methyl groups to fuel the methionine cycle. Where nitrate concentrations were higher, more GBT was partitioned for lipid biosynthesis by both bacteria and eukaryotic phytoplankton. Our data highlight unexpected metabolic pathways and potential routes of microbial metabolite exchange.