Abstract: The long-term excessive use of nitrogen (N) fertilizer in agricultural regions has increased soil nitrate and residual N accumulation and poses a threat to groundwater quality. Nitrate leaching into the vadose zone is becoming a global concern as the N stock in this habitat comprises a significant portion of N budgets. The vadose zone is also an essential channel for the conversion and reduction of nitrate. Therefore, the elimination of nitrate accumulation in the vadose zone is significant for maintaining groundwater safety. Microbial denitrification is the reduction of nitrogen nitrate (NO₃^--N) to gaseous nitric oxide (NO), nitrous oxide (N₂O), or dinitrogen (N₂). This mechanism is essential for removing excess nitrate in the subsoil before it leaches into the groundwater and saturates deep soil zones or discharges into ground aquifers through subsurface drainage. Therefore, isolating and screening bacteria with strong denitrification abilities may strengthen the vadose zone and aquifer microbial denitrification process, preventing groundwater nitrate pollution. In this study, 62 microbial denitrifiers were isolated from the 0-150 m vadose zone in a position experiment of long-term nitrogen application in the Agricultural Ecosystem Experimental Station of Luancheng, Chinese Academy of Sciences, located the North China Plain. 16S ribosomal RNA (16S rRNA) gene sequence analysis showed that the isolated denitrifiers had high homology with nine genera, belonging to the phyla Proteobacteria, Actinobacteria, and Firmicutes. Of the 62 denitrifiers, seven strains (L37, L71, L96, L103, L104, L133, and L13) were selected for denitrification potential experiments based on the phylogenetic tree results. Gas kinetics under anoxic incubations showed that three strains (L71, L13, and L103) could reduce nitrate substrates to nitrous oxides, such as N₂O and N₂, in anaerobic conditions. Electron microscopy showed that the denitrifying strains were 1.0 μm (L71), 1.5 μm (L13), and 1.5 μm (L103) long rod-shaped bacteria. Strain L103 had motile and complete denitrification abilities, and the denitrification rate was between 1.62 and 2.36 g(KNO₃)∙d^-1∙L^-1, indicating a high potential for use in agricultural practices. Furthermore, the denitrification ability of strain L103 was inhibited in acidic conditions, suggesting that pH also affects the microbial denitrification potential. Bacterial denitrifiers that reduce nitrate to N₂ in hypoxic/anoxic conditions exist in the deep vadose zone of the North China Plain. The denitrification potential of these strains is important for understanding how microorganisms contribute to the soil nitrate accumulation self-remediation process.