We aimed to investigate the temporal variation of in-stream net dissolved inorganic nitrogen (DIN) areal uptake rates (UDIN, in μg N m−2 min−1) and its implications on regulating catchment N export, under base flow conditions. To do so, we estimated UDIN from longitudinal profiles of ambient DIN concentration (nitrate + ammonium) in two streams on a monthly basis during two hydrological years (n = 45). We found that in-stream DIN uptake and release did not offset each other (UDIN ≠ 0) in half of the dates, and that UDIN > 0 occurred mostly in autumn. Based on these reach-scale uptake rates, we performed empirical calculations and model simulations to assess the potential of stream network DIN retention to regulate DIN export from catchments on an annual scale. The empirical approach consisted in up-scaling UDIN by means of a dynamic stream network analysis that considered temporal and spatial variation of UDIN. The modeling approach consisted in applying different scenarios with the INCA model based on the natural range of empirical UDIN values. Our results showed that the contribution of stream network DIN retention to catchment DIN export increased when calculations accounted for the temporal variation of UDIN. Both approaches suggested that stream network DIN retention can significantly reduce DIN export from headwater catchments under base flow conditions (from 4% to 38%).