River networks have the potential to permanently remove nitrogen through denitrification. Few studies have measured denitrification rates within an entire river network or assessed how land use affect rates at larger spatial scales. We sampled 108 sites throughout the network of the Fox River watershed, Wisconsin, to determine if land use influence sediment denitrification rates, and to identify zones of elevated sediment denitrification rates (hot spots) within the river network. Partial least squares regression models identified variables from four levels of organization (river bed sediment, water column, riparian zone, and watershed) that best predicted denitrification rates throughout the river network. Nitrate availability was the most important predictor of denitrification rates, while land cover was not always a good predictor of local-scale nitrate concentrations. Thus, land cover and denitrification rate were not strongly related across the Fox River watershed. A direct relationship between denitrification rate and watershed land cover occurred only in the Wolf River sub-watershed, the least anthropogenically disturbed of the sub-watersheds. Denitrification hot spots were located throughout the river network, regardless of watershed land use, with hot spot location being determined primarily by nitrate availability. In the Fox River watershed, when nitrate was abundant, river bed sediment character influenced denitrification rate, with higher denitrification rates at sites with fine, organic sediments. These findings suggest that denitrification occurring throughout an entire river network, from headwater streams to larger rivers, can help reduce nitrogen loads to downstream water bodies.