Intensifying agricultural production and coastal urbanization are increasing nitrogen (N) loads to estuaries, potentially increasing emissions of the greenhouse gas nitrous oxide (N2O). Here we present a first assessment of how changes in land use intensity affect estuarine N2O fluxes. We measured N2O concentrations over marine-freshwater transects in the wet and dry seasons in eight subtropical estuaries selected for differences in land use intensity. Daily estuary N loads ranged from 0.5 ± 0.4 kg N km−2 d−1 (minimally impacted) to 51 ± 30 kg N km−2 d−1 (highly impacted), corresponding to higher concentrations of all inorganic N species (nitrate, ammonium, and N2O) in the highly impacted estuaries. Net N2O fluxes from the eight estuaries ranged from −20 μg N2O-N m−2 d−1 (sink) to +300 μg N2O-N m−2 d−1 (source). However, neither N concentrations nor N loads explained the variations in N2O fluxes. Instead, seasonal differences in freshwater flushing times increased either N2O uptake (minimally impacted systems) or N2O efflux (moderately impacted systems) relative to N load. The lack of relationship between freshwater flushing times (kinetics) and N2O fluxes from the highly impacted estuaries, combined with evidence for both low carbon quality and phosphorous limitation in those systems, suggests that N2O emissions from highly impacted estuaries were controlled by stoichiometry rather than kinetics. This study shows that estuaries can shift from net sinks to sources of N2O as land use intensity increases but that the magnitude of this switch cannot be predicted based on N loads alone. (Figure presented.).