Ocean acidification in nitrogen-enriched estuaries has raised global concerns. For decades, biotic and abiotic denitrification in estuarine sediments has been regarded as the major ways to remove reactive nitrogen, but they occur at the expense of releasing greenhouse gas nitrous oxide (N₂O). However, how these pathways respond to acidification remains poorly understood. Here we performed a N₂O isotopocules analysis coupled with respiration inhibition and molecular approaches to investigate the impacts of acidification on bacterial, fungal, and chemo-denitrification, as well as N₂O emission, in estuarine sediments through a series of anoxic incubations. Results showed that acidification stimulated N₂O release from sediments, which was mainly mediated by the activity of bacterial denitrifiers, whereas in neutral environments, N₂O production was dominated by fungi. We also found that the contribution of chemo-denitrification to N₂O production cannot be ignored, but was not significantly affected by acidification. The mechanistic investigation further demonstrated that acidification changed the keystone taxa of sedimentary denitrifiers from N₂O-reducing to N₂O-producing ones and reduced microbial electron-transfer efficiency during denitrification. These findings provide novel insights into how acidification stimulates N₂O emission and modulates its pathways in estuarine sediments, and how it may contribute to the acceleration of global climate change in the Anthropocene.