Because　of　the　high　similarity　in　structure　and　sequence,　it　is　challenging　to　distinguish　the　S1　pocket　among　serine　proteases,　primarily　due　to　the　only　variability　at　residue　190　(A190　and　S190).　Peptide　or　protein-based　inhibitors　typically　target　the　negatively　charged　S1　pocket　using　lysine　or　arginine　as　the　P1　residue,　yet　neither　discriminates　between　the　two　S1　pocket　variants.　This　study　introduces　two　arginine　analogues,　L-4-guanidinophenylalanine　(12)　and　L-3-(N-amidino-4-piperidyl)alanine　(16),　as　novel　P1　residues　in　peptide　inhibitors.　16　notably　enhances　affinities　across　all　tested　proteases,　whereas　12　specifically　improved　affinities　towards　proteases　possessing　S190　in　the　S1　pocket.　By　crystallography　and　molecular　dynamics　simulations,　we　discovered　a　novel　mechanism　involving　a　water　exchange　channel　at　the　bottom　of　the　S1　pocket,　modulated　by　the　variation　of　residue　190.　Additionally,　the　specificity　of　12　towards　the　S190-presenting　S1　pocket　is　dependent　on　this　water　channel.　This　study　not　only　introduces　novel　P1　residues　to　engineer　inhibitory　potency　and　specificity　of　peptide　inhibitors　targeting　serine　proteases,　but　also　unveils　a　water-mediated　molecular　mechanism　of　targeting　serine　proteases.　©　2024　Elsevier　Inc.