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.