We used an in situ steady state 15N-labeled nitrate (15NO3−) and acetate (AcO−) well-to-wells injection experiment to determine how the availability of labile dissolved organic carbon (DOC) as AcO−influences microbial denitrification in the hyporheic zone of an upland (third-order) agricultural stream. The experimental wells receiving conservative (Cl− and Br) and reactive (15NO3−) solute tracers had hyporheic median residence times of 7.0 to 13.1 h, nominal flowpath lengths of 0.7 to 3.7 m, and hypoxic conditions (<1.5 mg O2 L−1). All receiving wells demonstrated 15N2 production during ambient conditions, indicating that the hyporheic zone was an environment with active denitrification. The subsequent addition of AcO− stimulated more denitrification as evidenced by significant δ15N2 increases by factors of 2.7 to 26.1 in receiving wells and significant decreases of NO3− and DO in the two wells most hydrologically connected to the injection. The rate of nitrate removal in the hyporheic zone increased from 218 kg ha−1 yr−1 to 521 kg ha−1 yr−1 under elevated AcO− conditions. In all receiving wells, increases of bromide and 15N2 occurred without concurrent increases in AcO−, indicating that 100% of AcO− was retained or lost in the hyporheic zone. These results support the hypothesis that denitrification in anaerobic portions of the hyporheic zone is limited by labile DOC supply.