We use a long‐term chemical and hydrologic record in combination with longitudinal sampling and high‐frequency nitrate (NO₃^‐) measurements from in situ sensors to describe temporal and spatial patterns of nitrogen (N) inputs and removal in the spring‐fed Ichetucknee River (Columbia County, Florida) and to determine the hydrological, geomorphic, and biological factors that influence those dynamics. Over a 20‐yr period of record, NO₃‐N removal averaged 118 kg N d^‐1 (0.77 g N m^‐2 d^‐1) over the upper 5 km of the Ichetucknee River. Three independent estimates of gross autotrophicNassimilation (from gross primary production, diel NO₃^‐ variation, and standing biomass) agreed closely but accounted for less than 20% of observed N removal. Longitudinal surveys indicate negligible or negative dissolved organic nitrogen and ammonium (NH₄⁺) production, suggesting that denitrification is the predominant mechanism of N removal in this river. A positive relationship between discharge and the magnitude of NO₃‐N removal shows that interactions with the surrounding floodplain exert considerable influence at high flows, and longitudinal NO₃^‐ patterns indicate that N removal may be influenced by channel morphology. These results suggest a greater role for dissimilatory processes and hydrologic connectivity with hyporheic and floodplain sediments than has been previously recognized in highly productive spring‐fed rivers of north Florida. While hydrologic variation is the primary determinant of variation in NO₃^‐ removal within the Ichetucknee River, comparison across systems indicates that biotic characteristics can cause significant deviation from predictions based on purely physical models of relationships between river size and N removal.