Severe　acute　respiratory　syndrome　coronavirus　2　(SARS-CoV-2)　is　the　viral　pathogen　causing　the　coronavirus　disease　2019　(COVID-19)　global　pandemic.　No　effective　treatment　for　COVID-19　has　been　established　yet.　The　serine　protease　transmembrane　protease　serine　2　(TMPRSS2)　is　essential　for　viral　spread　and　pathogenicity　by　facilitating　the　entry　of　SARS-CoV-2　into　host　cells.　The　protease　inhibitor　camostat,　an　anticoagulant　used　in　the　clinic,　has　potential　anti-inflammatory　and　antiviral　activities　against　COVID-19.　However,　the　potential　mechanisms　of　viral　resistance　and　antiviral　activity　of　camostat　are　unclear.　Herein,　we　demonstrate　high　inhibitory　potencies　of　camostat　for　a　panel　of　serine　proteases,　indicating　that　camostat　is　a　broad-spectrum　inhibitor　of　serine　proteases.　In　addition,　we　determined　the　crystal　structure　of　camostat　in　complex　with　a　serine　protease　(uPA　[urokinase-type　plasminogen　activator]),　which　reveals　that　camostat　is　inserted　in　the　S1　pocket　of　uPA　but　is　hydrolyzed　by　uPA,　and　the　cleaved　camostat　covalently　binds　to　Ser195.　We　also　generated　a　homology　model　of　the　structure　of　the　TMPRSS2　serine　protease　domain.　The　model　shows　that　camostat　uses　the　same　inhibitory　mechanism　to　inhibit　the　activity　of　TMPRSS2,　subsequently　preventing　SARS-CoV-2　spread.　IMPORTANCE　Serine　proteases　are　a　large　family　of　enzymes　critical　for　multiple　physiological　processes　and　proven　diagnostic　and　therapeutic　targets　in　several　clinical　indications.　The　serine　protease　transmembrane　protease　serine　2　(TMPRSS2)　was　recently　found　to　mediate　SARS-CoV-2　entry　into　the　host.　Camostat　mesylate　(FOY　305),　a　serine　protease　inhibitor　active　against　TMPRSS2　and　used　for　the　treatment　of　oral　squamous　cell　carcinoma　and　chronic　pancreatitis,　inhibits　SARS-CoV-2　infection　of　human　lung　cells.　However,　the　direct　inhibition　mechanism　of　camostat　mesylate　for　TMPRSS2　is　unclear.　Herein,　we　demonstrate　that　camostat　uses　the　same　inhibitory　mechanism　to　inhibit　the　activity　of　TMPRSS2　as　uPA,　subsequently　preventing　SARS-CoV-2　spread.