Tropical forests are important contributors for N₂O emissions from soil to the atmosphere; however, the microbial processes responsible for soil N₂O production in tropical forest soils remain unclear. In this study, we employed a 40-day-long in situ ¹⁵NO₃^- labeling experiment to determine the microbial processes that are responsible for N₂O production in an undisturbed tropical rainforest in Xishuangbanna, southwest China. We observed that during the experiment, most of the ¹⁵N-labeled NO₃^- were immobilized and only 0.027% was emitted as N₂O. Of the total N₂O production, 3.1% was from the added ¹⁵N-labeled NO₃^-, 48.6% was from the added NH₄⁺ and native N. Within the first 27 h, nitrification (heterotrophic and autotrophic nitrification) was the predominant microbial process of N₂O production, accounting for 97.6% of total soil N₂O emissions, whereas denitrification and co-denitrification resulted in only 1.6% and 0.9% emissions, respectively. To the best of our knowledge, this is the first study to demonstrate that nitrification, rather than denitrification, is the predominant microbial process of N₂O production in situ in the study area in the tropical forest.