Ruminant urine deposition onto pasture creates hot-spots where emissions of nitrous oxide (N2O) are produced by aerobic and anaerobic microbial pathways. However, limited measurements of in situ soil oxygen (O-2)-N2O relationships hinder the prediction of N2O emissions from urine-affected soil. This study tested whether soil O-2 concentration or relative diffusivity of O-2 (D-p/D-O) could explain N2O emissions from urine patches. Using a randomized plot design, N2O emissions were measured daily from a perennial ryegrass (Lolium perenne L.) pasture for 56 d following bovine (Bos taurus) urine deposition to an imperfectly drained silty loam soil. Soil O-2, volumetric water content, pH, conductivity, and extractable N and C were measured in urine-amended and non-amended soil. Values of water-filled pore space (WFPS) and D-P/D-O were modeled. When data from treatments were pooled together, daily mean D-P/D-O explained 73% of the total variance in mean daily N2O flux, compared with 65, < 60, and <20% for WFPS, O-2 and other measured variables, respectively. Soil pH, O-2, volumetric water content, WFPS and D-P/D-O all explained more of the variance in the urine-amended compared with the non-amended soil. Daily N2O fluxes increased substantially at D-P/D-O values around 0.006, which was consistent with past laboratory studies. These results demonstrate for the first time an O-2 diffusion threshold for elevated N2O fluxes in the field, expressed as D-P/D-O approximate to 0.006. Further studies should examine the consistency of this threshold under varying N and C substrates and a range of soil pH.