Widespread　use　of　nanomaterials　raises　concerns.　The　underlying　mechanism　by　which　graphene　oxide　(GO)　nanoparticles　causes　poor　settleability　of　activated　sludge　remains　unclear.　To　explore　this　mechanism,　three　reactors　with　different　GO　concentrations　were　established.　Extended　Derjaguin-Landau-Verwey-Overbeek　theory　indicated　that　GO　destroyed　the　property　of　extracellular　polymeric　substances　(EPS),　increasing　the　energy　barrier　between　bacteria.　Low　levels　of　uronic　acid　and　hydrogen　bonding　in　exopolysaccharide　weakened　the　EPS　gelation　increasing　aggregation　repulsion.　Lager　amounts　of　hydrophilic　amino　acid　and　looser　structure　of　extracellular　proteins　for　exposing　inner　hydrophilic　groups　significantly　contributed　to　the　hydrophilicity　of　EPS.　Both　changes　implied　deterioration　in　EPS　structure　under　GO　stress.　Metagenome　demonstrated　a　decrease　in　genes　responsible　for　capsular　polysaccharide　colonization　and　genes　regulated　the　translocation　of　loose　proteins　were　increased,　which　increased　repulsion　between　bacteria.　This　study　elucidated　that　changes　in　EPS　secretion　under　GO　exposure　are　the　underlying　causes　of　poor　settleability.　©　2024　Elsevier　Ltd