The oxygen isotopic composition of nitrate (NO₃⁻) produced by nitrification (δ¹⁸O_NO3,nit) establishes the initial isotopic signature of NO₃⁻ in environmental systems and provides a baseline for tracing NO₃⁻ sources and subsequent transformations. However, how δ¹⁸O_NO3,nit and its relationship with water δ¹⁸O (δ¹⁸O_H2O) vary with temperature and substrate availability in agricultural soils remains poorly constrained. Moreover, soil evaporation can enrich δ¹⁸O_H2O in surface soils where nitrification is most active, yet how this enrichment influences δ¹⁸O_NO3,nit under contrasting hydrological conditions remains unexplored. To address these gaps, we integrated soil incubation experiments with a synthetic transport modeling framework based on StorAge Selection (SAS) functions to quantify how biogeochemical and hydrological factors regulate the variability and predictability of δ¹⁸O_NO3,nit in an agricultural soil. The incubation experiments showed that, across wide ranges of temperature (7–35 °C) and ammonium (NH₄⁺) availability (0–98 mg N kg⁻¹ soil), δ¹⁸O_NO3,nit generally tracked soil δ¹⁸O_H2O but varied by 3.4–5.3 ‰ at a given δ¹⁸O_H2O. Temperature and NH₄⁺ availability influenced δ¹⁸O_NO3,nit primarily by regulating transient nitrite (NO₂⁻) accumulation, which enhanced NO₂⁻–water oxygen isotope exchange and thereby altered the expression of the associated equilibrium isotope effect. Synthetic transport modeling further revealed that hydrological processes can generate δ¹⁸O_NO3,nit variability comparable in magnitude to that produced by biogeochemical controls. Because δ¹⁸O_NO3,nit records the δ¹⁸O of water in nitrification zones at the time of NO₃⁻ production, whereas drainage δ¹⁸O_H2O is additionally shaped by the mixing of water transported through heterogeneous flow paths, using drainage δ¹⁸O_H2O to predict δ¹⁸O_NO3,nit can produce systematic biases, particularly when seasonal nitrification dynamics interact with soil evaporation and water transport. Overall, our results show that δ¹⁸O_NO3,nit in soil is jointly controlled by biogeochemical processes that regulate the expression of kinetic and equilibrium isotope effects during nitrification and by hydrological processes that determine the sources, ages, and flow paths of water supplying nitrification. These findings underscore the need to move beyond stream-centric approaches toward reactive transport frameworks that explicitly accounts for dynamic biogeochemical controls across diverse flow paths and water ages when applying NO₃⁻ isotopes to environmental systems.