Nanoplastics　(NPs)　are　ubiquitously　present　in　wastewater　treatment　plants,　which　would　be　removed　by　the　flocculation　of　extracellular　polymeric　substances　(EPS)　from　activated　sludge.　However,　the　interaction　mechanisms　between　NPs　and　EPS　of　activated　sludge　remain　largely　unexplored.　This　study　investigated　the　interaction　mechanisms　between　polystyrene　nanoplastics　(PS-NPs)　and　EPS　with　sodium　acetate　(NaAc),　methanol　(MeOH)　and　glucose　(GLC)　as　carbon　sources.　The　results　showed　that　the　functional　group　involved　in　the　interactions　between　PS-NPs　and　EPS　was　the　carbonyl　of　protein　amide　I　region.　The　interaction　between　PS-NPs　and　EPS　increased　the　β-sheets　content,　decreased　the　ratio　of　α-helix　to　(β-sheet　+　random　coil),　and　changed　the　protein　secondary　structures　to　strong　rigidity.　This　enhanced　the　flocculation　of　activated　sludge　cultivated　by　these　three　carbon　sources.　The　flocculation　between　PS-NPs　and　EPS　in　activated　sludge　using　NaAc　as　the　carbon　source　was　the　strongest　among　these　three　carbon　sources.　Therefore,　the　degree　of　flocculation　between　NPs　and　EPS　of　activated　sludge　in　wastewater　treatment　plants　varies　with　carbon　sources.　This　work　provides　a　reference　for　the　NPs　removal　mechanisms　from　wastewater,　which　will　help　to　understand　the　migration　behavior　of　MPs　and　NPs　in　wastewater　treatment　processes.　©　2022　Elsevier　Ltd