Subsoiling reduces N2O emissions by altering the relative gas diffusivity, O2 status and microbial communities in grazed pasture soil

• Subsoiling enhanced soil macroporosity, O 2 status and relative gas diffusivity at 0–20 depth. • Subsoiling reduced cumulative N 2 O emissions by 52% and 81% for non-urine and urine-treated plots, respectively. • Relative gas diffusivity explained 59–87% of N 2 O flux variation during the first 15 days after urine application. • Subsoiling decreased nirK gene abundance linked to denitrification activity. Nitrous oxide (N 2 O) is a potent greenhouse gas predominantly emitted from grazed pasture through denitrification, driven by soil oxygen (O 2 ) availability and urine-derived nitrogen (N). Pasture soils are vulnerable to compaction from animal treading, restricting gas diffusion and enhancing N 2 O emissions. Although subsoiling alleviates compaction, its impact on soil O 2 status and N 2 O emissions, particularly under high urine N load, remain poorly understood and rarely investigated. This in-situ field study (March-August 2023) evaluated the effect of subsoiling on soil moisture, O 2 content, relative gas diffusivity (D p /D o ), functional gene abundance, N 2 O emissions, and pasture production. Treatments included non-subsoiling or subsoiling, each with or without synthetic ruminant urine (713 kg N ha −1 ). Subsoiling improved macroporosity, enhanced O 2 availability, increased D p /D o at 5, 10 and 20 cm depth ( P < 0.001 ), and reduced moisture at 10 cm depth ( P < 0.001 ). Subsoiling significantly reduced N 2 O emissions by 52% and 81% of non-subsoiled plots for non-urine and urine treatments, respectively ( P < 0.05 ). D p /D o was strongly correlated with N 2 O fluxes during the first 15 days following urine application ( R 2 = 0.59 – 0.87 ), suggesting its utility as a predictive indicator under high substrate availability. Molecular analysis showed reduced nirK gene abundance under subsoiling, with limited response for other denitrification genes. Subsoiling had no significant effect on pasture yield or N uptake. Overall, subsoiling mitigates N 2 O emissions by improving soil aeration and D p /D o while maintaining productivity, offering a promising strategy for sustainable N management in grazed pasture soils.