Land use/land cover change often leads to increased nutrient loading to streams; however, its influence on stream ecosystem nutrient transport remains poorly understood. Given the deleterious impacts elevated nutrient loading can have on aquatic ecosystems, it is imperative to improve understanding of nutrient retention capacities across stream scales and watershed development gradients. We performed 17 nutrient addition experiments on six streams across the West Fork Gallatin Watershed, Montana, USA, to quantify nitrogen uptake kinetics and retention dynamics across stream sizes (first to fourth order) and along a watershed development gradient. We observed that stream nitrogen (N) uptake kinetics and spiraling parameters varied across streams of different development intensity and scale. In more developed watersheds we observed a fertilization affect. This fertilization affect was evident as increased ash-free dry mass, chlorophylla, and ambient and maximum uptake rates in developed as compared to undeveloped streams. Ash-free dry mass, chlorophylla, and the number of structures in a subwatershed were significantly correlated to nutrient spiraling and kinetic parameters, while ambient and average annual N concentrations were not. Additionally, increased maximum uptake capacities in developed streams contributed to low in-stream nutrient concentrations during the growing season, and helped maintain watershed export at low levels during base flow. Our results indicate that land use/land cover change can enhance in-stream uptake of limiting nutrients and highlight the need for improved understanding of the watershed dynamics that control nutrient export across scales and development intensities for mitigation and protection of aquatic ecosystems.