The heavy use of nitrogen fertilizer in intensive agricultural areas often leads to nitrate accumulation in subsurface soil and nitrate contamination in groundwater, which poses a serious risk to public health. Denitrifying microorganisms in the subsoil convert nitrate to gaseous forms of nitrogen, thereby mitigating the leaching of nitrate into groundwater. Here, we investigated denitrifying microorganisms in the deep vadose zone of a typical intensive agricultural area in China through microcosm enrichment, genome-resolved metagenomic analysis, and denitrifying bacteria isolation. A total of 1000 metagenome-assembled genomes (MAGs) were reconstructed, resulting in 98 high-quality, dereplicated MAGs that contained denitrification genes. Among them, 32 MAGs could not be taxonomically classified at the genus or species level, indicating that a broader spectrum of taxonomic groups is involved in subsoil denitrification than previously recognized. A denitrifier isolate library was constructed by using a strategy combining high-throughput and conventional cultivation techniques. Assessment of the denitrification characteristics of both the MAGs and isolates demonstrated the dominance of truncated denitrification. Functional screening revealed the highest denitrification activity in two complete denitrifiers belonging to the genus Pseudomonas. These findings greatly expand the current knowledge of the composition and function of denitrifying microorganisms in subsoils. The constructed isolate library provided the first pool of subsoil-denitrifying microorganisms that could facilitate the development of microbe-based technologies for nitrate attenuation in groundwater.