Quercetin-3-rutinoside　(rutin)　is　a　bioflavonoid　that　is　common　in　foods.　The　finding　that　quercetin-3-rutinoside　inhibits　protein　disulfide　isomerase　(PDI)　and　potently　blocks　thrombosis　in　vivo　has　enabled　the　evaluation　of　PDI　inhibition　in　multiple　animal　models　of　thrombus　formation　and　has　prompted　clinical　studies　of　PDI　inhibition　in　thrombosis.　Nonetheless,　how　quercetin-3-rutinoside　blocks　PDI　activity　remains　an　unanswered　question.　Combining　NMR　spectroscopy,　site-directed　mutagenesis,　and　biological　assays,　we　identified　H256　as　the　key　residue　for　PDI　interacting　with　quercetin-3-rutinoside.　Quercetin-3-rutinoside　inhibited　the　activity　of　PDI　(WT)　but　not　PDI　(H256A).　Molecular　dynamic　simulations　indicated　that　the　flavonoid　skeleton,　but　not　the　rutinoside　conjugate,　is　embedded　in　the　major　binding　pocket　on　the　b′　domain.　Among　several　quercetin-3-rutinoside　analogues　tested,　only　compounds　with　a　phenoxyl　group　at　position　7　showed　direct　binding　to　PDI,　further　supporting　our　molecular　model.　Studies　using　purified　coagulation　factors　showed　that　quercetin-3-rutinoside　inhibited　the　augmenting　effects　of　PDI　(WT),　but　not　PDI　(H256A),　on　tissue　factor　(TF)　activity.　Quercetin-3-rutinoside　also　inhibited　chemotherapy-induced　TF　activity　enhancement　on　endothelial　cells.　Together,　our　studies　show　that　residue　H256　in　PDI　and　the　phenoxyl　group　at　position　7　in　quercetin-3-rutinoside　are　essential　for　inhibition　of　PDI　by　quercetin-3-rutinoside.　These　results　provide　new　insight　into　the　molecular　mechanism　by　which　flavonoids　block　PDI　activity.　©　2022　American　Chemical　Society.　All　rights　reserved.