Human activities have increased the global pool of nitrogen (N) and changing land use may increase N reaching small streams. We examined anthropogenic impacts on N cycling in Midwestern streams draining subwatersheds dominated by forested, agricultural, or urban land use and focused on factors controlling sediment denitrification using the chloramphenicol-amended acetylene inhibition technique. Land use affected denitrification activity primarily via its effect on NO3- concentrations and sediment organic content. Forested streams had the lowest denitrification rates and the lowest NO3- and dissolved organic carbon concentrations, compared to agricultural and urban streams. At the landscape scale, forested streams removed a greater proportion of their NO3- load via denitrification compared to agricultural and urban streams. Factors controlling denitrification included sediment depth, substrata type, water source, and nutrient limitation. We should consider those factors when comparing denitrification rates across habitats, different methods, and when scaling denitrification rates for use in ecosystem modeling.