Abstract: Soil microorganisms are pivotal drivers of nitrogen (N) cycling, profoundly influencing crop productivity and ecosystem stability. While extensive research has focused on N transformations in surface soils, the mechanisms governing deep soil N cycling, particularly across diagnostic horizons of Isohumosols (Chernozems), remain poorly understood. This study aimed to elucidate the vertical distribution patterns of N-cycling functional genes in Ustic (semi-arid) and Udic (humid) Isohumosols and identify the key environmental drivers regulating these processes. Soil samples were collected from four soil profiles encompassing three diagnostic horizons — humus (A), illuviation (B), and parent material (C) layers. Real-time qPCR was employed to quantify six key functional genes: nitrification markers (Comammox, AOA amoA, AOB amoA) and denitrification markers (nirK, nirS, nosZ). Potential nitrification (PNR) and denitrification rates (PDR) were measured, and relationships with soil physicochemical properties were analyzed using redundancy analysis (RDA) and structural equation modeling (SEM). The results revealed a pronounced depth-dependent decline in gene abundances, with 41.8~96.8% reductions across diagnostic horizons (P<0.05). Notably, Udic Isohumosols exhibited distinct N-cycling gene profiles compared to Ustic counterparts. In the A horizon, Udic soils showed 49.7%, 89.7%, 30.4%, and 18.8% higher abundances of nosZ, nirS, nirK, and Comammox genes, respectively (P<0.05), but 64.2% and 20.1% lower AOA amoA and AOB amoA abundances. These differences diminished in B and C horizons except for Comammox and AOA amoA. Correspondingly, PNR and PDR decreased by 53.7% and 25.5% from A to B horizons (P<0.05), with Udic soils displaying 26.6~71.9% lower PNR across all horizons but only 7.9% reduced PDR in the A layer. Strong positive correlations were observed between PNR and nitrification genes (R²=0.82, P<0.001) and between PDR and denitrification genes (R²=0.34, P<0.001), confirming functional gene abundance as a robust predictor of N-cycling potential. Innovatively, this study identified available phosphorus (AP) and pH as primary regulators of N-cycling processes. AP exhibited significant positive correlations with Comammox, AOB amoA, nirK, nirS, and nosZ abundances (P<0.001), while pH negatively influenced comammox and nirS but positively correlated with AOB amoA (P<0.05). RDA and SEM analyses demonstrated that AP and pH collectively explained 61% and 21% of variations in PNR and PDR, respectively. AP regulated nitrification indirectly by modulating gene abundances (path coefficient=−0.21, P<0.05), whereas pH exerted direct effects on both nitrifiers and denitrifiers. The SEM further highlighted that AP’s total effect on PDR surpassed that of denitrification genes, underscoring its pivotal role in N metabolism. Additional factors, including soil organic carbon (SOC), C:N ratio, clay content, and ammonium/nitrate levels, contributed to microhabitat differentiation—clay and specific surface area influenced oxygen and moisture gradients, while SOC and C:N shaped substrate availability for microbial communities. Two key innovations emerge from this work: First, it provides the first comprehensive evidence of contrasting N-cycling strategies between Ustic and Udic Isohumosols, linked to moisture-driven redox conditions. Udic soils favored comammox and denitrifiers in surface layers, likely due to chronic waterlogging and NH₄⁺ accumulation, while Ustic soils supported ammonia oxidizers under drier, higher-pH conditions. Second, the identification of AP (rather than traditional N substrates) as a master variable redefines understanding of nutrient coupling in Isohumosols, suggesting phosphorus availability critically constrains microbial N transformations across soil profiles. These findings advance the mechanistic framework for predicting N-cycling dynamics in deep soils and offer actionable insights for optimizing fertilization strategies. By highlighting AP management and pH adjustment as potential levers to mitigate N losses or enhance N retention, this study establishes a scientific basis for sustainable soil management in Isohumosol-dominated agroecosystems. Future research should explore how long-term phosphorus amendment interacts with moisture regimes to shape N-cycling microbial networks at the genome-resolved level.