Purpose The excessive use of nitrogen (N) fertilizer in intensive agriculture has increased nitrate leaching into groundwater, but its impacts on N transformation processes and the associated microbial communities in the deep vadose zone remain unclear. Materials and methods Soil samples from 0-1050 cm depth were collected from a 20-year field experiment with two N fertilization treatments: 0 (N0) and 600 kg N ha(-1) year(-1) (N600). Amplicon sequencing and quantitative PCR analyses were performed to profile the vertical distribution of soil microbial communities and denitrification genes. Results and discussion The soil microbial community structure and diversity were strongly influenced by soil depth and N fertilization. The 250 cm depth was identified as a threshold depth, as dramatically different microbial communities were found below and above this depth. Quantitative PCR results showed that the absolute abundance of denitrification genes decreased with increasing soil depth. Conclusion This study elucidated the profound effects of long-term N input on the composition and diversity of the microbial communities and the abundance of denitrifiers in the deep vadose zone. Our results provide basic information for use in mitigating nitrate leaching by enhancing microbial denitrification in deep vadose zones in intensive agricultural areas.