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Abstract Agricultural activities are the major anthropogenic source of nitrous oxide (N2O), an important greenhouse gas and ozone-depleting substance. However, the role of forage conservation as a potential source of N2O has rarely been studied. We investigated N2O production from the simulated silage of the three major crops—maize, alfalfa, and sorghum—used for silage in the United States, which comprises over 90% of the total silage production. Our findings revealed that a substantial N2O could be generated, potentially placing forage conservation as the third largest N2O source in the agricultural sector. Notably, the application of chlorate as an additive significantly reduced N2O production, but neither acetylene nor intermittent exposure to oxygen showed any impact. Overall, the results highlight that denitrifiers, rather than nitrifiers, are responsible for N2O production from silage, which was confirmed by molecular analyses. Our study reveals a previously unexplored source of N2O and provides a crucial mechanistic understanding for effective mitigation strategies.