Natural environmental isotopes of nitrate and ammonium are used in conjunction with major ion chemistry and hydrological data to establish controls upon the biogeochemical cycling of nitrogen beneath a High Arctic polythermal glacier (Midtre Lovénbreen). Here, high nitrate concentrations in subglacial meltwaters suggest that the subglacial environment may be furnishing nitrate in excess of that released from the snowpack and glacier ice. Isotopic values of δ18ONO3 suggest the provenance of such excess nitrate to be microbial in origin and δ15NNO3 indicates the source nitrogen compounds to have high δ15N values relative to supraglacial runoff. We address the nitrification of supraglacial ammonium, the mineralization of organic nitrogen and the oxidation of geologic ammonium as potential sources of this additional nitrate. Mass fluxes of N compounds in a subglacial river and their δ15N ratios indicate that the nitrification of supraglacial ammonium delivered to the glacier bed can account for much, but not all, of the excess nitrate. The additional source most likely involves the mineralization of organic nitrogen, although δ15N values in rock samples suggest that the dissolution of rock-derived ammonium cannot be discounted if large fractionation effects occur during dissolution. Our results therefore agree with previous catchment scale mass balance studies at the site, which report a major internal loss of NH4+ from the snowpack following melt. However, at the catchment scale, the NH4+ loss is greater than the excess of NO3− observed in runoff, indicating that microbial assimilation of ammonia into organic matter in a range of other habitats is also likely. The identification of NH4+assimilation and nitrification further highlights the non-conservative behaviour of nitrogen in glacial environments and testifies to the importance of microbially-mediated reactions in the biogeochemical cycling of nitrogen in an environment that has, until recently, been regarded as biologically inert.