To investigate the effects of temperature shock on N₂O emissions, four treatments with rapidly changing incubation temperature from the control (20 °C) to 4, 12, 25, or 34 °C were conducted. Results showed that higher N₂O emissions (0.023–0.37%) were observed when reactor contents received temperature shocks. N₂O emissions increased as the temperature interval increased. Nitrate, nitrite, and nitrous oxide reduction rates generally followed the order: 34 °C > 25 °C > 20 °C > 12 °C > 4°C. Overall, the low-temperature shocks down-regulated and high-temperature shocks up-regulated the expression of denitrifying genes. However, the transcription rate of norB/nosZ and nirS/nosZ could not explain higher N₂O emission. The increased N₂O emissions might be more related to post-transcriptional regulation and enzyme activity (Q₁₀ value). The results of cDNA sequencing showed that the active microbial community was relatively stable. Among the members of top 15 genera with active transcripts, Flavobacterium, Comamonadaceae and Xanthomonadales were the dominant denitrifying bacteria.