The Mississippi River and other large rivers have the potential to regulate nitrogen export from terrestrial landscapes, and thus mitigate eutrophication in downstream aquatic ecosystems. In large rivers, human-constructed impoundments and connected backwaters may facilitate nitrogen removal; however, the capacity of these features is poorly quantified and incompletely incorporated into model frameworks. Using a high-resolution and spatially intensive sampling technique, we assessed the contribution of individual navigation pools, as well as impounded open waters and backwater wetlands within them, to overall nitrate retention by mapping the entire length (1370 km) of the Upper Mississippi River (UMR) main channel. Based on this single spatial survey of water chemistry, the river appeared to act primarily as a passive nitrate transporter, retaining only 12.5% of the incoming load, most of which occurred in the upper 150 km of the river, which includes the largest and only naturally impounded reach of the river. Although reservoirs typically are nitrogen sinks, our data indicate that UMR dams do not impede river flows to the extent necessary to promote substantial changes in water residence times and subsequent nitrogen removal. Backwaters routinely had lower nitrate concentrations than the main channel, but their limited hydrologic connectivity to the through-flowing river channel constrained their influence on downstream export. As a whole, the UMR did not remove a substantial proportion of its nitrate load despite optimal N removal conditions, numerous impoundments, and the presence of extensive backwater habitats. These results suggest that efforts to reduce delivery of nitrogen to the Gulf of Mexico should emphasize mitigation strategies that target upland nutrient sources rather than relying on removal within the Mississippi River.