Denitrification is a major process contributing to the removal of nitrogen (N) from ecosystems, but its rate is difficult to quantify. The natural abundance of isotopes can be used to identify the occurrence of denitrification and has recently been used to quantify denitrification rates at the ecosystem level. However, the technique requires an understanding of the isotopic enrichment factor associated with denitrification, which few studies have investigated in forest soils. Here, soils collected from two tropical and two temperate forests in China were incubated under anaerobic or aerobic laboratory conditions for two weeks to determine the N and oxygen (O) isotope enrichment factors during denitrification. We found that at room temperature (20℃),NO₃^- was reduced at a rate of 0.17 to 0.35μg N g^-1 h^-1, accompanied by the isotope fractionation of N (¹⁵ε) and O (¹⁸ε) of 31‰ to 65‰ (48.3±2.0‰ on average) and 11‰ to 39‰ (18.9±1.7‰ on average), respectively. The N isotope effects were, unexpectedly, much higher than reported in the literature for heterotrophic denitrification (typically ranging from 5‰ to 30‰) and in other environmental settings (e.g., groundwater, marine sediments and agricultural soils). In addition, the ratios of Δδ¹⁸O:Δδ¹⁵N ranged from 0.28 to 0.60 (0.38±0.02 on average), which were lower than the canonical ratios of 0.5 to 1 for denitrification reported in other terrestrial and freshwater systems. We suggest that the isotope effects of denitrification for soils may vary greatly among regions and soil types and that gaseous N losses may have been overestimated for terrestrial ecosystems in previous studies in which lower fractionation factors were applied.