The aim of our research was to obtain information on the isotopic fingerprint of nitrous oxide (N2O) associated with its production and consumption during denitrification. An arable soil was preincubated at high moisture content and subsequently amended with glucose (400 kg C ha−1) and KNO3 (80 kg N ha−1) and kept at 85% water-filled pore space. Twelve replicate samples of the soil were incubated for 13 days under a helium-oxygen atmosphere, simultaneously measuring gas fluxes (N2O, N2 and CO2) and isotope signatures (δ18O-N2O, δ15Nbulk-N2O, δ15Nα, δ15Nβ and 15N site preference) of emitted N2O. The maximum N2O flux (6.9 ± 1.8 kg N ha−1 day−1) occurred 3 days after amendment application, followed by the maximum N2 flux on day 4 (6.6 ± 3.0 kg N ha−1 day−1). The δ15Nbulk was initially −34.4‰ and increased to +4.5‰ during the periods of maximum N2 flux, demonstrating fractionation during N2O reduction, and then decreased. The δ18O-N2O also increased, peaking with the maximum N2 flux and remaining stable afterwards. The site preference (SP) decreased from the initial +7.5 to −2.1‰ when the N2O flux peaked, and then simultaneously increased with the appearance of the N2 peak to +8.6‰ and remained stable thereafter, even when the O2 supply was removed. We suggest that this results from a non-homogenous distribution of NO in the soil, possibly linked to the KNO3 amendments to the soil, causing the creation of several NO pools, which affected differently the isotopic signature of N2O and the N2O and N2 fluxes during the various stages of the process. The N2O isotopologue values reflected the temporal patterns observed in N2O and N2 fluxes. A concurrent increase in 15N site preference and δ18O of N2O was found to be indicative of N2O reduction to N2.