A dual-isotope and solute flux mass-balance was used to elucidate the processes that lead to attenuation of nitrogen contamination in an agriculturally impacted river. The River Wensum drains a lowland catchment with an area of 570 km² in East Anglia, eastern England. Analysis of nitrate concentration, δ¹⁵N(NO₃) and δ¹⁸O(NO₃) of samples from the River Wensum collected from upstream locations to the catchment outlet through all seasons and flow conditions showed a consistent pattern of increasing isotope values with decreasing nitrate concentrations downstream. δ¹⁵N(NO₃) and δ¹⁸O(NO₃) of catchment surface water and groundwater samples revealed a dominant influence from microbially cycled and nitrified source-nitrogen, which results in high nitrate concentrations in Chalk groundwater and upstream in the River Wensum. Denitrification of Chalk groundwater-baseflow in the hyporheic zone results in the downstream trend observed in the river. Hyporheic denitrification is estimated to remove 931 kg/day of nitrate-nitrogen by the catchment outlet, representing 31% of the potential riverine nitrate load. The use of dual-isotope and solute flux modeling at the catchment scale is a novel application to quantify denitrification within the river valley, demonstrating the importance of hyporheic zone processes in attenuating the impacts of anthropogenic contamination of hydrologic systems.