The Guadalquivir Estuary is the largest estuary in the southwest basin of the Iberian Peninsula, which is subject to strong anthropogenic influence such as the damming or the multitude of crop fields on its margins. Nitrous Oxide (N2O) variability is analysed considering the influence of temperature, salinity, water-atmosphere fluxes, benthic fluxes, reactivity and lateral inputs. N2O increases along the salinity gradient, with values ranging from 5.9 to 103.3 nmol L−1. Thus, values of N2O concentration are very close to equilibrium with the atmosphere at the mouth, while in the inner zone the fluxes to the atmosphere are higher, showing the greatest variability of N2O in the estuary (74.26 ± 7.41 μmol m−2 d−1). Sediments act as a source of N2O to the water column, with benthic fluxes presenting a wide range from 2 to 20 μmol m−2 d−1. Denitrification processes in the sediments may be important in the inner part of the estuary, where negative benthic fluxes of nitrate have been observed. Production rates of N2O in the water column are estimated from incubation experiments, resulting in higher production with temperature, and lower with salinity. Lateral inputs are calculated by balance of the different processes characterized and seems to be an important factor influencing N2O variability in the inner zone of the estuary.