Streams play a critical role in attenuating the excess reactive nitrogen generated from human activities. These systems can consequently also emit significant amounts of N2O, a potent greenhouse gas. Models and manipulative experiments now suggest that hydrology regulates the balance between nitrogen removal and N2O production. We aimed to empirically test this hypothesis by measuring changes in the concentration and isotopic composition of NO3- (δ18O, δ15N) and N2O (δ18O, δ15N, site preference) in hyporheic sediments and surface water of a 30 m reach over eight days of falling stream discharge (2.7 to 1.8 m3 s-1). The stream was persistently heterotrophic (productivity/respiration: 0.005 - 0.2), while changes in conductivity, δ18O-H2O, and 222Rn indicated that hyporheic mixing decreased and net groundwater inputs increased as discharge declined. The shallow groundwater had high inorganic N concentrations (2 – 10 mg l-1), but increased groundwater inputs could not fully explain the concurrent increases in NO3- (1 – 3 mg N l-1) and N2O (700 to 1000 % saturation) in the surface water. Biologically, rather than solely hydrologically, regulated stream nitrogen export was confirmed by changes in N2O and NO3- isotopic composition. However, isotope patterns indicated that nitrification, not denitrification, increased surface water NO3- and N2O concentrations as hyporheic exchange decreased. These findings empirically demonstrate how flow dynamics regulate biological NO3- production as well as transport, with implications for predicting aquatic N2O emissions.