Extensively grazed grasslands are understudied in terms of their contribution to greenhouse gas (GHG) emissions from livestock production. Mountains, moorlands and heath occupy 18% of the UK land area, however, in situ studies providing high frequency N2O emissions from sheep urine deposited to such areas are lacking. Organic soils typical of these regions may provide substrates for denitrification-related N2O emissions, however, acidic and anoxic conditions may inhibit nitrification (and associated emissions from nitrification and denitrification). We hypothesised urine N2O-N emission factors (EFs) would be lower than the UK country-specific and IPCC default value for urine, which is based on lowland measurements. Using automated GHG sampling chambers, N2O emissions were determined from real sheep urine (930 kg N ha−1) and artificial urine (920 kg N ha−1) applied in summer, and from an artificial urine treatment (1120 kg N ha−1) and a combined NO3− and glucose treatment (106 kg N ha−1; 213 kg C ha−1) in autumn. The latter treatment provided an assessment of the soils capacity for denitrification under non-substrate limiting conditions. The artificial urine-N2O EF was 0.01 ± 0.00% of the N applied in summer and 0.00 ± 0.00% of the N applied in autumn. The N2O EF for real sheep urine applied in summer was 0.01 ± 0.02%. A higher flux was observed in only one replicate of the real urine treatment, relating to one chamber where an increase in soil solution NO3− was observed. No lag phase in N2O emission was evident following application of the NO3− and glucose treatment, which emitted 0.69 ± 0.15% of the N applied.This indicates nitrification rates are the bottle-neck for N2O emissions in upland organic soils. We calculated the potential impact of using hill-grazing specific urine N2O EFs on the UK inventory of N2O emissions from sheep excreta, and found a reduction of ca. 43% in comparison to the use of a country-specific excretal EF.