Mountainous watersheds are characterized by variability in functional traits, including vegetation, topography, geology, and geomorphology, which determine nitrogen (N) retention, and release. Coal Creek and East River are two contrasting catchments within the Upper Colorado River Basin that differ markedly in total nitrate (NO3−) export. The East River has a diverse vegetation cover, and sinuous floodplains, and is underlain by N-rich marine shale. At 0.21 ± 0.14 kg ha−1 yr−1, the East River exports ∼3.5 times more NO3− relative to the conifer-dominated Coal Creek (0.06 ± 0.02 kg ha−1 yr−1). While this can partly be explained by the larger size of the East River, the distinct watershed traits of these two catchments imply different mechanisms controlling the aggregate N-export signal. A causality analysis shows physical and biogenic processes were critical in determining NO3− export from the East River catchment. Stable isotope ratios of NO3− (δ15NNO3 and δ18ONO3) show the East River catchment is a strong hotspot for biogeochemical processing of NO3− at the hillslope soil-saprolite. By contrast, the conifer-dominated Coal Creek retained nearly all atmospherically deposited NO3−, and its export was controlled by catchment hydrological traits (i.e., snowmelt periods and water table depth). The conservative N-cycle within Coal Creek is likely due to the abundance of conifer trees, and smaller riparian regions, retaining more NO3− overall and reduced processing prior to export. This study highlights the value of integrating isotope systematics to link watershed functional traits to mechanisms of watershed element retention and release.