In transitional environments, the role of sediments biogeochemistry and denitrification is crucial for establishing their buffer potential against nitrate (NO3−) pollution. The Po River (Northern Italy) is a worldwide hotspot of eutrophication. However, benthic N dynamics and the relevance of denitrification in its delta have not yet been described. The aim of the present study was to quantify the contribution of denitrification in attenuating the NO3− loading transported to the sea during summer. Benthic fluxes of dissolved inorganic nitrogen (N) and denitrification rates were measured in laboratory incubations of intact sediment cores collected, along a salinity gradient, at three sections of the Po di Goro, the southernmost arm of the Po Delta. The correlation between NO3− consumption and N2 production rates demonstrated that denitrification was the main process responsible for reactive N removal. Denitrification was stimulated by both NO3− availability in the Po River water and organic enrichment of sediment likely determined by salinity-induced flocculation of particulate organic load, and inhibited by increasing salinity, along the river–sea gradient. Overall, denitrification represented a sink of approximately 30% of the daily N loading transported in middle summer, highlighting a previously underestimated role of the Po River Delta.