AbstractAcid-sulphate sugarcane soils in the subtropics are known hot-spots for nitrous oxide (N₂O) emissions, yet the reduction of reactive N₂O to non-reactive dinitrogen (N₂) via specific pathways remains a major uncertainty for nitrogen (N) cycling and loss from these soils. This study investigated the magnitude and the N₂O:N₂ partitioning of N₂O and N₂ losses from a subtropical acid-sulphate soil under sugarcane production using the ¹⁵N gas flux method, establishing the contribution of hybrid (co- and chemo-denitrification) and heterotrophic denitrification to N₂O and N₂ losses. Soils were fertilised with potassium nitrate, equivalent to 25 and 50 kg N ha⁻¹, watered close to saturation then incubated over 30 days. An innovative, fully automated incubation system coupled to an isotope-ratio mass-spectrometer enabled real time analysis of ¹⁵N₂O and ¹⁵N₂ at sub-diel resolution. Peak losses of N₂O and N₂ reached 6.5 kg N ha⁻¹ day⁻¹, totalling > 50 kg of N₂O+N₂-N ha⁻¹. Emissions were dominated by N₂, accounting for more than 57% of N₂O+N₂ losses, demonstrating that the reduction of N₂O to N₂ proceeded even under highly acidic conditions. Over 40% of N₂O, but only 2% of N₂ emissions, were produced via hybrid pathways. These findings demonstrate hybrid pathways are generally limited to N₂O production, likely driven by high organic matter content and low soil pH, promoting both biotic, and abiotic nitrosation. Regardless of the underlying process, the magnitude of the N₂O emissions demonstrates the environmental, but also the potential agronomic significance, of hybrid pathways of N₂O formation for N loss from fertilised acid-sulphate soils.