Biogeochemical reactions associated with stream nitrogen cycling, such as nitrification and denitrification, can be strongly controlled by water and solute residence times in the hyporheic zone (HZ). We used a whole-stream steady state 15N-labeled nitrate (15NO3−) and conservative tracer (Cl−) addition to investigate the spatial and temporal physiochemical conditions controlling the denitrification dynamics in the HZ of an upland agricultural stream. We measured solute concentrations (15NO3−, 15N2 (g), as well as NO3−, NH3, DOC, DO, Cl−), and hydraulic transport parameters (head, flow rates, flow paths, and residence time distributions) of the reach and along HZ flow paths of an instrumented gravel bar. HZ exchange was observed across the entire gravel bar (i.e., in all wells) with flow path lengths up to 4.2 m and corresponding median residence times greater than 28.5 h. The HZ transitioned from a net nitrification environment at its head (short residence times) to a net denitrification environment at its tail (long residence times). NO3− increased at short residence times from 0.32 to 0.54 mg-N L−1 until a threshold of 6.9 h and then consistently decreased from 0.54 to 0.03 mg-N L−1. Along these same flow paths, declines were seen in DO (from 8.31 to 0.59 mg-O2 L−1) and DOC (from 3.0 to 1.7 mg-C L−1). The rates of the DO and DOC removal and net nitrification were greatest during short residence times, while the rate of denitrification was greatest at long residence times. 15NO3− tracing confirmed that a fraction of the NO3− removal was via denitrification as 15N2 was produced across the entire gravel bar HZ. Production of 15N2 across all observed flow paths and residence times indicated that denitrification microsites are present even where nitrification was the net outcome. These findings demonstrate that the HZ is an active nitrogen sink in this system and that the distinction between net nitrification and denitrification in the HZ is a function of residence time and exhibits threshold behavior. Consequently, incorporation of HZ exchange and water residence time characterizations will improve mechanistic predictions of nitrogen cycling in streams.