Nafamostat　mesylate　(NM)　is　a　synthetic　compound　that　inhibits　various　serine　proteases　produced　during　the　coagulation　cascade　and　inflammation.　Previous　studies　showed　that　NM　was　a　highly　safe　drug　for　the　treatment　of　different　cancers,　but　the　precise　functions　and　mechanisms　of　NM　are　not　clear.　In　this　study,　we　determined　a　series　of　crystal　structures　of　NM　and　its　hydrolysates　in　complex　with　a　serine　protease　(urokinase-type　plasminogen　activator　[uPA]).　These　structures　reveal　that　NM　was　cleaved　by　uPA　and　that　a　hydrolyzed　product　(4-guanidinobenzoic　acid　[GBA])　remained　covalently　linked　to　Ser195　of　uPA,　and　the　other　hydrolyzed　product　(6-amidino-2-naphthol　[6A2N])　released　from　uPA.　Strikingly,　in　the　inactive　uPA　(uPA-S195A):NM　structure,　the　6A2N　side　of　intact　NM　binds　to　the　specific　pocket　of　uPA.　Molecular　dynamics　simulations　and　end-point　binding　free-energy　calculations　show　that　the　conf1　of　NM　(6A2N　as　P1　group)　in　the　uPA-S195A:NM　complex　may　be　more　stable　than　conf2　of　NM　(GBA　as　P1　group).　Moreover,　in　the　structure　of　uPA:NM　complex,　the　imidazole　group　of　His57　flips　further　away　from　Ser195　and　disrupts　the　stable　canonical　catalytic　triad　conformation.　These　results　not　only　reveal　the　inhibitory　mechanism　of　NM　as　an　efficient　serine　protease　inhibitor　but　also　might　provide　the　structural　basis　for　the　further　development　of　serine　protease　inhibitors.