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 μM) 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.