Protein　disulfide　isomerase　(PDI)　is　a　vital　oxidoreductase.　Extracellular　PDI　promotes　thrombus　formation　but　does　not　affect　physiological　blood　hemostasis.　Inhibition　of　extracellular　PDI　has　been　demonstrated　as　a　promising　strategy　for　antithrombotic　treatment.　Herein,　we　focused　on　the　major　substrate　binding　site,　a　unique　pocket　in　the　PDI　b　＇　domain,　and　identified　four　natural　products　binding　to　PDI　by　combining　virtual　screening　with　tryptophan　fluorescence-based　assays　against　a　customized　natural　product　library.　These　hits　all　directly　bound　to　the　PDI-b　＇　domain　and　inhibited　the　reductase　activity　of　PDI.　Among　them,　galangin　showed　the　most　prominent　potency　(5.9　mu　M)　against　PDI　and　as　a　broad-spectrum　inhibitor　for　vascular　thiol　isomerases.　In　vivo　studies　manifested　that　galangin　delayed　the　time　of　blood　vessel　occlusion　in　an　electricity-induced　mouse　thrombosis　model.　Molecular　docking　and　dynamics　simulation　further　revealed　that　the　hydroxyl-substituted　benzopyrone　moiety　of　galangin　deeply　inserted　into　the　interface　between　the　PDI-b　＇　substrate-binding　pocket　and　the　a　＇　domain.　Together,　these　findings　provide　a　potential　antithrombotic　drug　candidate　and　demonstrate　that　the　PDI　b　＇　domain　is　a　critical　domain　for　inhibitor　development.　Besides,　we　also　report　an　innovative　high-throughput　screening　method　for　the　rapid　discovery　of　PDI　b　＇　targeted　inhibitors.