Global　estuarine　ecosystems　are　experiencing　severe　nitrogen　pollution　and　ocean　acidification　(OA)　simultaneously.　Sedimentary　denitrification　is　an　important　way　of　reactive　nitrogen　removal　but　at　the　same　time　leads　to　the　emission　of　large　amounts　of　nitrous　oxide　(N2O),　a　potent　greenhouse　gas.　It　is　known　that　OA　in　estuarine　regions　could　impact　denitrification　and　N2O　production;　however,　the　underlying　mechanism　is　still　underexplored.　Here,　sediment　incubation　and　pure　culture　experiments　were　conducted　to　explore　the　OA　impacts　on　microbial　denitrification　and　the　associated　N2O　emissions　in　estuarine　sediments.　Under　neutral　(in　situ)　conditions,　fungal　N2O　emission　dominated　in　the　sediment,　while　the　bacterial　and　fungal　sources　had　a　similar　role　under　acidification.　This　indicated　that　acidification　decreased　the　sedimentary　fungal　denitrification　and　likely　inhibited　the　activity　of　fungal　denitrifiers.　To　explore　molecular　mechanisms,　a　denitrifying　fungal　strain　of　Penicillium　janthinellum　was　isolated　from　the　sediments.　By　using　deuterium-labeled　single-cell　Raman　spectroscopy　and　isobaric　tags　for　relative　and　absolute　quantitation　proteomics,　we　found　that　acidification　inhibited　electron　transfers　in　P.　janthinellum　and　downregulated　expressions　of　the　proteins　related　to　energy　production　and　conservation.　Two　collaborative　pathways　of　energy　generation　in　the　P.　janthinellum　were　further　revealed,　that　is,　aerobic　oxidative　phosphorylation　and　TCA　cycle　and　anoxic　pyruvate　fermentation.　This　indicated　a　distinct　energy　supply　strategy　from　bacterial　denitrification.　Our　study　provides　insights　into　fungi-mediated　nitrogen　cycle　in　acidifying　aquatic　ecosystems.　©　2022　American　Chemical　Society.