Symbiotic relationships between N 2 -fixing prokaryotes and their autotrophic hosts are essential in nitrogen (N)-limited ecosystems, yet the importance of this association in pristine boreal peatlands, which store 25 % of the world's soil (C), has been overlooked. External inputs of N to bogs are predominantly atmospheric, and given that regions of boreal Canada anchor some of the lowest rates found globally (*1 kg N ha -1 year -1), biomass production is thought to be limited primarily by N. Despite historically low N deposition, we show that boreal bogs have accumulated approximately 12-25 times more N than can be explained by atmospheric inputs.Here we demonstrate high rates of biological N 2 -fixation in prokaryotes associated with Sphagnum mosses that can fully account for the missing input of N needed to sustain high rates of C sequestration. Additionally, N amendment experiments in the field did not increase Sphagnum production, indicating that mosses are not limited by N. Lastly, by examining the composition and abundance of N 2 -fixing prokaryotes by quantifying gene expression of 16S rRNA and nitrogenase-encoding nifH, we show that rates of N 2 -fixation are driven by the substantial contribution from methanotrophs, and not from cyanobacteria. We conclude biological N 2 -fixation drives high sequestration of C in pristine peatlands, and may play an important role in moderating fluxes of methane, one of the most important greenhouse gases produced in peatlands. Understanding the mechanistic controls on biological N 2 -fixation is crucial for assessing the fate Responsible Editor: Matthew Wallenstein.Electronic supplementary material The online version of this article (doi:10.1007/s10533-014-0019-6) contains supplementary material, which is available to authorized users. Biogeochemistry (2014) 121:317-328 DOI 10.1007 of peatland carbon stocks under scenarios of climate change and enhanced anthropogenic N deposition.