Zebra mussels (Dreissena polymorpha) have altered the structure of invaded ecosystems and exhibit characteristics that suggest they may influence ecosystem processes such as nitrogen (N) cycling. We measured denitrification rates seasonally on sediments underlying zebra mussel beds collected from the impounded zone of Navigation Pool 8 of the Upper Mississippi River. Denitrification assays were amended with nutrients to characterize variation in nutrient limitation of denitrification in the presence or absence of zebra mussels. Denitrification rates at zebra mussel sites were high relative to sites without zebra mussels in February 2004 (repeated measures analysis of variance (RM ANOVA), p = 0.005), potentially because of high NO3-N variability from nitrification of high NH4+ zebra mussel waste. Denitrification rates were highest in June 2003 (RM ANOVA, p < 0.001), corresponding with the highest NO3-N concentrations during the study (linear regression, R2 = 0.72, p < 0.001). Denitrification was always N-limited, but sites without zebra mussels showed the strongest response to N amendments relative to sites with zebra mussels (two-way ANOVA, p ≤ 0.01). Examining how zebra mussels influence denitrification rates will aid in developing a more complete understanding of the impact of zebra mussels and more effective management strategies of eutrophic waters.