[Background] N2O is a powerful greenhouse gas with a 265-fold stronger warming potential than CO2. Fertilization plays an important role in affecting N2O emission from soils driven by bacterial community, and denitrification is the major source of N2O under anaerobic conditions. [Objective] To investigate N2O emission and greenhouse soil bacterial community response to the overuse of nitrogen fertilizer. [Methods] Robot system was used to monitor the denitrifying gas (N2O and N2) kinetics of soils during anaerobic incubation, and compare the difference of N2O emissions between traditional fertilization and reduced nitrogen fertilization. The soil microbial community structure was analyzed by sequencing the 16S rRNA gene V3?V4 region using Illumina MiSeq. [Results] The nitrate concentration in conventional nitrogen fertilization soil (CNS) was about two folds higher than that in reduced nitrogen fertilization soil (RNS). CNS showed higher N2O accumulation and emission rate during earlier anaerobic-incubation stage although the nitrate content was adjusted to the same level in both types of soil. Traditional fertilization significantly changed the bacterial community structure, and decreased the microbial diversity. Although Rhodanobacter was the most abundant genus both in CNS and RNS, it was enriched by traditional fertilization. However, relative abundance of denitrifying functional genes (narG, nirK, norB, nosZ) had little response to the overuse of fertilizer. [Conclusion] Traditional fertilization reshaped the bacterial community in soil. Overuse nitrogen fertilizer influenced N2O emission from soil via changing the microbial community including the microbiota related with nitrogen transformations.