Agricultural nonpoint source pollution from the upper areas of the Upper Mississippi, Missouri, and Ohio River basins accounts for the majority of the excess nitrogen that leads to the Gulf of Mexico's hypoxic zone. However, agriculture landscapes across the USA, Europe, and China are undergoing major changes related to the proliferation of confined animal feeding operations (CAFOs) that account for a greater proportion of point source contamination. Mitigating long-term nutrient inputs at a large scale (Mississippi River Basin) requires understanding these microscale changes at the small watershed level (less than 100km2). To assess the control of land-use and subsurface hydrological processes on nutrient fate and transport, temporal patterns in nutrient concentrations in a mixed land-use karstic watershed were analyzed. To assess the control of differential land-use sources on total inorganic nitrogen (TIN) loads within the watershed, 4months of weekly water sampling was performed at a spring location and at two underground conduit locations. The observed temporal variations in nutrient concentrations are primarily associated with seasonal changes in land-use associated with corn growth. Data show that land-use sources explain much of the temporal variability of TIN at the spring when weighted against the hydrological factor. End-member-mixing analysis of dissolved organic carbon (DOC) shows a progressive increase in the contribution of DOC-enriched sources and a more labile form of carbon toward the harvest time. Overall, during high flow, nonpoint source infiltration from manure-fertilized croplands in the northern branch (NB) dominate DOC loads. Because conduit-dominated karstic aquifers are more susceptible to contamination from direct and fast (< 7h) subsurface infiltration during late summer rainfall period (July to August), a slight advance in the timing of manure application could substantially reduce nutrient loads to local groundwater. A combined evaluation of subsurface hydrological processes and land-use factors controlling nutrients at the scale of small watersheds is crucial to developing site-specific nutrient management plans and managing the Gulf of Mexico's hypoxic zone.