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.