Sediments in the backwaters of the Upper Mississippi River (UMR) are highly organic and provide an optimal environment for N removal. We monitored an 8.6-ha UMR backwater site near La Crosse, Wisconsin, for nearly 3 y to assess temporal variability, seasonal trends, and the factors regulating denitrification. We measured rates of unamended denitrification (DEN) and denitrification enzyme activity (DEA) rates at ambient temperature and DEA at 30°C (DEA30). Seasonal mean (±1 SE) DEN rates ranged from 0.041 ± 0.015 to 0.47 ± 0.23 μg N cm⁻² h⁻¹ and were highest in winter and lowest in autumn. Seasonal rates of DEA exhibited a different pattern with the highest rates in summer (25.6 ± 3.4 μg N cm⁻² h⁻¹) and the lowest rates in winter (10.6 ± 2.1 μg N cm⁻² h⁻¹). The overall mean DEA30 rate was 31.0 ± 1.9 μg N cm⁻² h⁻¹ but showed no significant seasonal pattern. Short-term (weekly) and seasonal variability exhibited by rates of DEN and DEA were best explained by water-column NO₃⁻ concentration and temperature, respectively. No environmental variables explained a significant amount of variability in DEA30. Our results suggest that nutrient (i.e., NO₃⁻) availability and temperature are both regulators of denitrification, with NO₃⁻ concentration being the most important limiting factor in this system. The high DEN rates during winter were in response to elevated NO₃⁻ concentrations resulting from a chain reaction beginning with algal blooms creating oxic conditions that stimulated nitrification. Increasing hydrological connectivity in large rivers as a river management tool to reduce N flux to downstream areas may be beneficial.