Investigating how the abundance of denitrifying functional genes nirS, nirK, nosZ I and nosZ II respond to temperature and nitrogen addition in upland Ultisol can provide guidance for agricultural nutrient management and environmental protection in this region. In this study, soils were sampled from a long-term fertilization experiment and used for a microcosm incubation experiment under the conditions of three nitrogen addition treatments: N 0, 25, and 50 mg/kg, and three temperature levels at 15, 25, and 35 °C. Soils were incubated in the dark and destructively sampled on days 7 and 30 of incubation. After sampling, soil DNA was extracted, and the abundances of denitrifying functional genes were determined by real-time quantitative PCR. Results showed that the abundances of nirS, nirK, nosZ I and nosZ II genes were the highest at 25 ℃ after 7-day incubation. However, after 30-day incubation, the abundances of nirS, nirK, nosZ I and nosZ II genes were the highest at 15 ℃ and were decreased with increasing temperature. Moreover, nitrogen addition had no significant effect on the abundances of all the denitrifying functional genes. In addition, three-way ANOVA showed that the interactions of temperature, nitrogen addition and incubation time significantly influenced the abundances of denitrifying functional genes. Overall, the abundances of denitrifying functional genes are substantially influenced by temperature but less affected by the nitrogen addition. The abundances of denitrifying functional genes may vary considerably on both a daily and seasonal basis, and this should be taken into consideration during soil sampling and nitrous oxide emission measuring.