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 δ ¹⁸O-to-δ ¹⁵N differed between soils, ranging from 1.6 to 2.7. By contrast, during N₂O reduction, we observed a constant ratio of δ ¹⁸O-to-δ ¹⁵N 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.