Protein　disulfide　isomerase　(PDI)　is　a　founding　member　of　the　thiol　isomerase　family,　and　is　recently　found　to　play　critical　roles　in　thrombus　formation.　The　development　of　effective　PDI　inhibitors　is　of　great　significance,　and　attracts　strong　interest.　We　previously　showed　that　rutin　bound　directly　to　PDI　and　inhibited　PDI　activities,　leading　to　the　suppression　of　platelet　aggregation　and　fibrin　generation　in　a　mouse　model.　A　close　analog　of　rutin,　isoquercetin,　is　currently　in　advanced　phase　clinical　trials.　However,　the　molecular　interaction　between　rutin　and　PDI　is　unknown　and　is　difficult　to　study　by　X-ray　crystallography　due　to　the　weak　interaction.　Here,　we　generated　a　molecular　model　of　PDI:rutin　complex　by　molecular　docking　and　thorough　molecular　dynamics　(MD)　simulations.　We　then　validated　the　complex　model　through　a　number　of　different　experimental　methods.　We　mutated　the　key　residues　predicted　by　the　model　and　analyzed　the　mutants　by　an　optimized　isothermal　titration　calorimetry　(ITC)　method　and　a　functional　assay　(insulin　reduction　assay).　The　results　consistently　showed　that　the　PDI　residues　H354,　L355　and　E359　are　important　in　the　binding　of　rutin.　These　residues　are　next　to　the　canonical　major　substrate　binding　site　of　the　b　domain,　and　were　not　conserved　across　the　members　of　thiol　isomerases,　explaining　the　specificity　of　rutin　for　PDI　among　vascular　thiol　isomerases.　Furthermore,　the　inhibitory　activities　of　three　rutin　analogues　were　evaluated　using　an　insulin　reduction　assay.　The　results　supported　that　the　second　sugar　ring　at　the　side　chain　of　rutin　was　not　necessary　for　the　binding　to　PDI.　Together,　this　work　provides　the　structural　basis　for　the　inhibitory　mechanism　of　rutin　to　PDI,　and　offers　a　promising　strategy　for　the　design　of　new　generation　inhibitors　with　higher　binding　affinity　to　PDI　for　therapeutic　applications.