Globally, approximately 50% of nitrogen (N) fertilizer applied in agricultural practices escapes into the environment, resulting in water and air pollution and ozone depletion. Soil N transformation processes determine the chemical form of N and the amount of the various N forms, controlling where N fertilizer goes and how much of it is lost. Therefore, a comprehensive understanding of soil N transformation responses to N fertilizer is critical for developing effective management strategies to improve soil N retention capacity and minimize soil N losses. Using 15N tracing techniques with acetylene inhibition, three ranges for ammonium concentration in fertilizer were identifed (I: 14–16, II: 46–59, and III: 90–115 mg N kg− 1, depending on type of organic materials added) that determine competition for mineral N among N transformation processes under increased carbon availability in an agricultural soil. Increasing ammonium concentration caused a shift from a nitrate assimilation-dominated (<I) to an equally ammonium assimilation- and nitrifcation-dominated (I-II) to nitrifcation-dominated (II-III), and fnally to nitrifcation-predominant (>III) period in organic amendment addition treatments. Structural equation modeling revealed that ammonium addition inhibited soil nitrate assimilation by enhancing nitrifcation and ammonium assimilation, resulting in the shift. Consequently, the ratios of nitrifcation to ammonium assimilation and to gross N assimilation (nitrate assimilation + ammonium assimilation) increased signifcantly in response to elevated ammonium concentration with organic amendment addition, indicating lower N retention capacity and higher potential risks of N loss. Overall, we present a comprehensive picture of how concurrent gross N transformation processes interact to compete for mineral N in response to ammonium-forming fertilizer application, and demonstrate that nitrifcation and ammonium assimilation weaken the stimulating effect of organic amendment on nitrate assimilation with increasing ammonium concentration.