Nitrification inhibitors (NIs) have been shown to reduce emissions of the greenhouse gas nitrous oxide (N₂O) from agricultural soils. However, their N₂O reduction efficacy varies widely across different agro-ecosystems, and underlying mechanisms remain poorly understood. To investigate effects of the NI 3,4-dimethylpyrazole-phosphate (DMPP) on N-turnover from a pasture and a horticultural soil, we combined the quantification of N₂ and N₂O emissions with ¹⁵N tracing analysis and the quantification of the N₂O-reductase gene (nosZ) in a soil microcosm study. Nitrogen fertilization suppressed nosZ abundance in both soils, showing that high nitrate availability and the preferential reduction of nitrate over N₂O is responsible for large pulses of N₂O after the fertilization of agricultural soils. DMPP attenuated this effect only in the horticultural soil, reducing nitrification while increasing nosZ abundance. DMPP reduced N₂O emissions from the horticultural soil by >50% but did not affect overall N₂ + N₂O losses, demonstrating the shift in the N₂O:N₂ ratio towards N₂ as a key mechanism of N₂O mitigation by NIs. Under non-limiting NO₃⁻ availability, the efficacy of NIs to mitigate N₂O emissions therefore depends on their ability to reduce the suppression of the N₂O reductase by high NO₃⁻ concentrations in the soil, enabling complete denitrification to N₂.