In mitigating agricultural nitrous oxide (N₂O) emissions, partially substituting chemical nitrogen (N) fertilizers with organic amendments is widely advocated for; however, the effectiveness of this approach varies, and the optimal substitution ratio remains unclear. This study employed a long-term field experiment in a maize cropping system, integrating in situ N₂O flux monitoring, soil analysis, and metagenomic sequencing to investigate the effects of substituting synthetic N fertilizer with sheep manure at different ratios (one-sixth, one-third, and full substitution) on cumulative N₂O emissions and underlying microbial mechanisms. The results showed that substituting one-third of chemical N with sheep manure (OF2) most effectively reduced cumulative N₂O emissions, by 18.64% (2021) and 47.00% (2022), compared to conventional chemical fertilization (NPK). This treatment optimized key soil properties, significantly lowering soil nitrate (NO₃⁻-N) and ammonium (NH₄⁺-N) concentrations while increasing soil organic matter. Metagenomic analysis revealed that the mitigation was primarily driven by a coordinated shift in the nitrogen-cycling microbial functional gene network: OF2 significantly enhanced the abundance of N₂O reductase genes (nosZ clades I and II) and increased the nosZ/(nirK + nirS) gene ratio, thereby strengthening the genetic potential for complete denitrification (N₂O → N₂). Partial least squares path modeling confirmed that soil properties indirectly reduced N₂O emissions, primarily by regulating the microbial community and functional gene abundances. This study provides mechanistic evidence that partial organic substitution (with one-third sheep manure) mitigates N₂O emissions by optimizing the soil habitat and reprogramming the functional gene network of nitrogen-cycling microbes, with a key emphasis on enhancing the N₂O reduction pathway.