Despite advancements in fertilizer management practices, such as applying crop-specific doses, splitting fertilizer applications throughout the growing season, and selecting appropriate types of fertilizers, global estimates suggest that agroecosystems lose approximately half of the applied nitrogen fertilizer. This inefficiency arises partly because the potential for spatial precision application is not fully utilized. Additionally, research on further strategies to improve nitrogen use efficiency remains limited. The environmental consequences of these nitrogen losses, including gaseous emissions and leaching, are a significant concern, contributing to ecosystem degradation and climate change. Soil nitrogen losses can be mitigated through practices that increase organic carbon inputs, enhance native soil organic carbon, and, most importantly, boost soil microbial biomass levels. However, there is a limited understanding of how soil-plant interactions influence nitrogen immobilization and plant uptake across gradients of organic carbon inputs, native soil organic carbon levels, and microbial biomass. To address this knowledge gap, our study aims to determine the fate of fertilizer nitrogen within a plant-soil system under varying organic matter quantity and quality. To this end, we conducted a field experiment within a long-term organic amendment trial spanning over 45 years. This trial is characterized by a gradient in soil organic carbon and microbial biomass, induced by differing rates of repeated manure amendments. To introduce short-term organic carbon input variability, we further established a gradient in fresh organic carbon using harvest residues. Microplots were installed along these gradients and fertilized with 140 kg N/ha of nitrogen-15-enriched ammonium nitrate, applied in three split doses, to trace the fate of fertilizer-derived nitrogen. We are assessing its incorporation into wheat grain, straw, root biomass, and soil pools, including the microbial and organic nitrogen pools. Preliminary data indicate that neither microbial biomass nor soil organic carbon directly affected the uptake of fertilizer nitrogen into the aboveground plant biomass (grain and straw). However, we hypothesize that, at the plant-soil level, fertilizer recovery and, therefore, nitrogen use efficiency will improve with higher organic carbon inputs, greater native soil organic carbon, and more abundant microbial biomass. This improvement primarily being driven by enhanced nitrogen immobilization within the soil. Through this research, we aim to elucidate the connections between carbon and nitrogen cycling, with a particular focus on the role of soil carbon-to-nitrogen stoichiometry in determining the fate of fertilized nitrogen in agricultural systems. Ultimately, our findings will contribute to the development of optimized residue management strategies to increase nitrogen use efficiency at the agroecosystem level without compromising yields and food security.