Agricultural watersheds in the Upper Mississippi River Basin (UMRB) face persistent challenges in nutrient loss reduction and water quality mitigation. Addressing water quality issues in this region requires a deep understanding of hydrological and biogeochemical processes from surface to subsurface. Groundwater is critical in hydrological processes and nitrogen export, yet its contributions remain insufficiently quantified due to the limitations in obtaining real-time observations. This research investigates the role of groundwater in a representative HUC8 agricultural watershed within the UMRB using three up-to-date process-based models with different structures in subsurface hydrological and biogeochemical processes: default SWAT+, SWAT+gwflow, and SWAT+-MODFLOW-RT3D. We assessed groundwater’s influence on hydrological and nitrogen cycling processes by integrating groundwater flow and reactive transport processes. Our results demonstrate that groundwater significantly impacts hydrological fluxes and nitrogen transport pathways. Specifically, groundwater sustains baseflow during dry periods and acts as a long-term nitrogen reservoir, exacerbating legacy effects that complicate managing nutrient loss reduction in the agricultural landscape. Besides, enhanced modeling approaches, which incorporate groundwater flow and reactive transport processes, improve the accuracy of water quality assessments, offering crucial insights into local nutrient dynamics. This study underscores the importance of groundwater in understanding water quality dynamics and managing nutrient pollution. The findings provide a theoretical foundation that helps guide nutrient management and conservation decisions, supporting long-term sustainability in agricultural watersheds in the Upper Mississippi River Basin.