Measuring the stable isotope composition of nitrous oxide (N2O) evolved from soil could improve our understanding of the relative contributions of the main microbial processes (nitrification and denitrification) responsible for N2O formation in soil. However, interpretation of the isotopic data in N2O is complicated by the lack of knowledge of fractionation parameters by different microbial processes responsible for N2O production and consumption. Here we report isotopic enrichment for both nitrogen and oxygen isotopes in two stages of denitrification, N2O production and N2O reduction. We found that during both N2O production and reduction, enrichments were higher for oxygen than nitrogen. For both elements, enrichments were larger for N2O production stage than for N2O reduction. During gross N2O production, the ratio of δ18O-to-δ15N differed between soils, ranging from 1.6 to 2.7. By contrast, during N2O reduction, we observed a constant ratio of δ18O-to-δ15N with a value near 2.5. If general, this ratio could be used to estimate the proportion of N2O being reduced in the soil before escaping into the atmosphere. Because N2O-reductase enriches N2O in both isotopes, the global reduction of N2O consumption by soil may contribute to the globally observed isotopic depletion of atmospheric N2O.