The capacity of a lake to remove reactive nitrogen (N) through denitrification has important implications both for the lake and for downstream ecosystems. In large oligotropic lakes such as Lake Superior, where nitrate (NO₃ ⁻) concentrations have increased steadily over the past century, deep oxygen penetration into sediments may limit the denitrification rates. We tested the hypothesis that the position of the redox gradient in lake sediments affects denitrification by measuring net N-fluxes across the sediment–water interface for intact sediment cores collected across a range of sediment oxycline values from nearshore and offshore sites in Lake Superior, as well as sites in Lake Huron and Lake Erie. Across this redox gradient, as the thickness of the oxygenated sediment layer increased from Lake Erie to Lake Superior, fluxes of NH₄ ⁺ and N₂ out of the sediment decreased, and sediments shifted from a net sink to a net source of NO₃ ⁻. Denitrification of NO₃ ⁻ from overlying water decreased with thickness of the oxygenated sediment layer. Our results indicate that, unlike sediments from Lake Erie and Lake Huron, Lake Superior sediments do not remove significant amounts of water column NO₃ ⁻ through denitrification, likely as a result of the thick oxygenated sediment layer.