Nitrogen inputs and microbial nitrogen cycling were investigated along a 79 km transect into the Greenland Ice Sheet (GrIS) during the main ablation season in summer 2010. The depletion of dissolved nitrate and production of ammonium (relative to icemelt) in cryoconite holes on Leverett Glacier, within 7.5 km of the ice sheet margin, suggested microbial uptake and ammonification respectively. Positive in situ acetylene assays indicated nitrogen fixation both in a debris-rich 100 m marginal zone and up to 5.7 km upslope on Leverett Glacier (with rates up to 16.3 μmoles C2H4 m−2 day−1). No positive acetylene assays were detected > 5.7 km into the ablation zone of the ice sheet. Potential nitrogen fixation only occurred when concentrations of dissolved and sediment-bound inorganic nitrogen were undetectable. Estimates of nitrogen fluxes onto the transect suggest that nitrogen fixation is likely of minor importance to the overall nitrogen budget of Leverett Glacier and of negligible importance to the nitrogen budget on the main ice sheet itself. Nitrogen fixation is however potentially important as a source of nitrogen to microbial communities in the debris-rich marginal zone close to the terminus of the glacier, where nitrogen fixation may aid the colonization of subglacial and moraine-derived debris.