<p>Assessing nitrogen (N) mass balance and use efficiency is essential for tracking progress toward sustainable agriculture. However, the links between N surpluses or deficits, crop productivity, and environmental N losses remain poorly understood, particularly in rotational cropping systems. In this study, we combined natural abundance N isotope measurements across soil, water, and crop N pools with laboratory denitrification experiments to quantify N source-sink strengths in a tile-drained corn-soybean field in Illinois, USA. These isotope data were incorporated into a dual mass and isotope balance model to constrain the relationships among denitrification, soybean biological N fixation (BNF), and changes in soil organic N (SON), and to link partial and complete N balances. We found that N isotopes exhibited distinct distributions across the soil–water-crop continuum that were sensitive to crop rotation but unresponsive to split fertilizer application. Across a range of plausible scenarios, estimated denitrification losses were comparable in magnitude to nitrate leaching losses. Over the corn-soybean rotation, maintaining a stable SON stock requires soybean BNF rates sufficient to offset substantial N removal in soybean grain. Relative to corn residue, soybean residue appears to contribute little to SON formation, and its rapid decomposition can promote environmental N losses during periods of low crop N demand. Overall, this study demonstrates that integrating isotopic evidence with mass balance approaches can reveal the processes regulating N cycling in the soil–water-crop system and constrain agroecosystem responses to crop rotation and management practices.</p>