Excessive nitrogen (N) fertilization in agricultural ecosystems strongly affects microbial N-cycling processes in soil. However, a comprehensive understanding of how microorganisms involved in each N transformation process respond to long-term N input is lacking. Here, using metagenomic sequencing combined with the direct assembly of N-cycling genes, we found that long-term N fertilization elevated the abundance of the microorganisms involved in most N-transforming processes but decre ased that of N-fixing assemblages. The composition of the microbial groups involved in each N-transforming process was altered by fertilization, even though the abundance of several functional genes was not significantly changed. The relative abundance of microbial genera participating in the same step of N-cycling processes correlated with different soil properties, suggesting niche separation of these N-cycling taxa. Our results also indicated that the different responses to N fertilization exhibited by the taxa within the same functional group may be important for sustaining microbial nitrogen cycling in complex and dynamic environments.