Subsurface wastewater infiltration system (SWIS) is important source of N2O emission, biological denitrification process relies on organic carbon source as electron donor, enabling denitrifying reductase (NAR, NIR, NOR, and N2OR) to reduce nitrate nitrogen to N2. However, freeze-thaw cycles cause changes in soil structure through physical and biological processes that increase organic matter availability and microbial activity, thereby accelerating upper (aerobic) organic matter degradation. This leads to carbon deficiency in the lower (anaerobic) layer, which reduces denitrification efficiency increasing N2O production and emissions. This study investigated effects of influent C/N ratios on N2O emissions from SWIS under freeze-thaw stress, and changes in denitrifying reductase and microbial communities. Results showed that as influent C/N ratio increased from 6:1–10:1, average TN removal of SWIS decreased from 89.59 % to 84.95 %, while N2O emission rate decreased from 0.106 mg·m−2·h−1 to 0.0333 mg·m−2·h−1, a reduction of 68.61 %. Meanwhile, N2OR activity increased by 29.95 % with C/N ratio increasing from 6 to 10, showing a significant reduction of N2O. High-throughput sequencing results revealed that Proteobacteria and Firmicutes showed a linear relationship with influent C/N. As C/N increased from 6 to 8 and 10, Proteobacteria relative abundance increased from 22.24 % ± 1.79–32.51 % ± 2.51 % and 39.59 % ± 3.45 %, while Firmicutes relative abundance decreased from 31.73 % ± 7.87–3.34 % ± 0.27 % and 2.19 % ± 0.07 %. These results indicated that sufficient supply of organic carbon provided abundant electron donors for denitrifying reductases in freeze-thaw-affected SWIS, promoting influent NO3--N reduction and decreasing N2O release rates. Conversely, under low C/N ratio conditions, electron acceptance capacity of N2OR was limited and N2OR activity was inhibited, resulting in N2O accumulation and emission. SynopsisThis paper shows how increasing the organic carbon supply in SWIS can reduce N2O emissions and improve nitrogen removal under freeze-thaw stress.