Water-saving irrigation strategies can successfully alleviate methane emissions from rice fields, but significantly stimulate nitrous oxide (N₂O) emissions because of variations in soil oxygen level and redox potential.  However, the relationship linking soil N₂O emissions to nitrogen functional genes during various fertilization treatments in water-saving paddy fields has rarely been investigated.  Furthermore, the mitigation potential of organic fertilizer substitution on N₂O emissions and the microbial mechanism in rice fields must be further elucidated.  Our study examined how soil N₂O emissions were affected by related functional microorganisms (ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), nirS, nirK and nosZ) to various fertilization treatments in a rice field in southeast China over two years.  In this study, three fertilization regimes were applied to rice cultivation: a no nitrogen (N) (Control), an inorganic N (N_i), and an inorganic N with partial N substitution with organic manure (N_i+N_o).  Over two rice-growing seasons, cumulative N₂O emissions averaged 0.47, 4.62 and 4.08 kg ha⁻¹ for the Control, N_i and N_i+N_o treatments, respectively.  In comparison to the N_i treatment, the N_i+N_o fertilization regime considerably reduced soil N₂O emissions by 11.6% while maintaining rice yield, with a lower N₂O emission factor (EF) from fertilizer N of 0.95%.  Nitrogen fertilization considerably raised the AOB, nirS, nirK and nosZ gene abundances, in comparison to the Control treatment.  Moreover, the substitution of organic manure for inorganic N fertilizer significantly decreased AOB and nirS gene abundances and increased nosZ gene abundance.  The AOB responded to N fertilization more sensitively than the AOA.  Total N₂O emissions significantly correlated positively with AOB and nirS gene abundances while having a negative correlation with nosZ gene abundance and the nosZ/nirS ratio across N-fertilized plots.  In summary, we conclude that organic manure substitution for inorganic N fertilizer decreased soil N₂O emissions primarily by changing the soil NO₃⁻-N, pH and DOC levels, thus inhibiting the activities of ammonia oxidation in nitrification and nitrite reduction in denitrification, and strengthening N₂O reduction in denitrification from water-saving rice paddies.