Archaea have been detected throughout the oceanic water column and are quantitatively important members of picoplankton in the deep ocean. Two common groups, group I Crenarchaeota and group II Euryarchaeota, are consistently detected in warm hydrothermal fluid and are assumed to have been drawn into the subseafloor, mixed with hydrothermal fluid and then expelled. However, because they remain resistant to cultivation, very little is known about their physiology. Here we show that cold deep-seawater from the axial valley of Endeavour Segment on the Juan de Fuca Ridge contains not only groups I and II archaea as expected, but also unique potentially archaeal nitrogenase (nifH) genes, which are required for nitrogen fixation. These nifH genes are phylogenetically distinct and have dissimilar G+C content compared with those of hydrothermal vent archaea, suggesting that they belong to non-thermophilic deep-sea archaea. Furthermore, this sample did not contain mcrA genes, which are present in methanogens, the only known archaeal nitrogen fixers. These nifH genes were not detected in upper water column samples, or in a deep-seawater sample 100 km away from the spreading axis of the Juan de Fuca Ridge. We propose that these unique nifH genes may be localized to archaea that circulate through the nitrogen-poor subseafloor at the mid-ocean ridge as part of their life cycle.