Background <p>The reduction of N₂O to N₂, catalyzed by two kinds of N₂O-reducing bacteria, is the only known biological pathway of N₂O sink. However, the distribution and function of N₂O-reducing bacteria in estuarine waters remains unknown. This study investigated the distribution and community characteristics of two clades of N₂O-reducing bacteria (clades I and II) and their potential function in the waters of the Pearl River Estuary (PRE).</p> Results <p>Our results reveal that the gene abundance of the NosZ-II type of N₂O-reducing bacteria was remarkably higher than the NosZ-I type, specifically 2.29 to 16.41 times higher in summer and 1.63 to 16.68 times higher in winter, indicating that the NosZ-II type should play a major role in reducing N₂O content of the waters of the PRE. Furthermore, higher <i>nos</i>Z I/<i>nir</i> and <i>nos</i>Z II/<i>nir</i> ratios were strongly associated with lower ΔN₂O concentration. The potential rate of N₂O reduction exhibited a gradual increase from upstream to downstream. High-throughput sequencing analysis revealed significant differences in the spatial distribution in the N₂O-reducing bacterial community from upstream to downstream, with Alphaproteobacteria and Bacteroidetes identified as the dominant types of NosZ-I and NosZ-II N₂O-reducing bacteria, respectively. The community composition of two types of N₂O-reducing bacteria was both influenced by NO₂⁻ concentration in summer. Additionally, co-occurrence analysis demonstrated that 76.67 and 95.45% of the connections between the two clades in summer and winter were positive, indicating a synergistic effect of two types of N₂O-reducing bacteria for N₂O reduction.</p> Conclusion <p>This study provides novel insights into the ecological distribution and functional potential of NosZ-I and NosZ-II clades of N₂O-reducing bacteria in the PRE, revealing their distinct yet complementary roles in mediating N₂O reduction processes within estuarine waters.</p>