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The Mesozoic is marked by several widespread occurrences of intense organic matter burial. Sediments from the largest of these events, the Cenomanian–Turonian Oceanic Anoxic Event (OAE 2) are characterized by lower nitrogen isotope ratios than are seen in modern marine settings. It has remained a challenge to describe a nitrogen cycle that could achieve such isotopic depletion. Here we use nitrogen-isotope ratios of porphyrins to show that eukaryotes contributed the quantitative majority of export production throughout OAE 2, whereas cyanobacteria contributed on average approximately 20%. Such data require that any explanation for the OAE nitrogen cycle and its isotopic values be consistent with a eukaryote-dominated ecosystem. Our results agree with models that suggest the OAEs were high-productivity events, supported by vigorous upwelling. Upwelling of anoxic deep waters would have supplied reduced N species (i.e., NH4+) to primary producers. We propose that new production during OAE 2 primarily was driven by direct NH4+-assimilation supplemented by diazotrophy, whereas chemocline denitrification and anammox quantitatively consumed NO3- and NO2-. A marine nitrogen reservoir dominated by NH4+, in combination with known kinetic isotope effects, could lead to eukaryotic biomass depleted in 15N.