Respiratory viruses, such as SARS-CoV-2 and influenza, exploit host proteases like TMPRSS2 44 for entry, making TMPRSS2 a prime antiviral target. Here, we report the identification and 45 characterization of Trypstatin, a 61-amino acid Kunitz-type protease inhibitor derived from 46 human hemofiltrate. Trypstatin inhibits TMPRSS2 and related proteases, with IC50 values in 47 the nanomolar range, comparable to the small molecule inhibitor camostat mesylate. In vitro 48 assays demonstrated that Trypstatin effectively blocks spike-driven entry of SARS-CoV-2, 49 SARS-CoV-1, MERS-CoV, and hCoV-NL63, as well as hemagglutinin-mediated entry of 50 influenza A and B viruses. In primary human airway epithelial cultures, Trypstatin significantly 51 reduced SARS-CoV-2 replication and retained activity in the presence of airway mucus. In 52 vivo, intranasal administration of Trypstatin to SARS-CoV-2-infected Syrian hamsters reduced 53 viral titers and alleviated clinical symptoms. These findings highlight Trypstatin’s potential as 54 a broad-spectrum antiviral agent against TMPRSS2-dependent respiratory viruses