uPA (urokinase-type plasminogen activator) is a potential therapeutic target in a variety of pathological conditions, including cancer. In order to find new principles for inhibiting uPA in murine cancer models, we screened a phage-displayed peptide library with murine uPA as bait. We thereby isolated several murine uPA-binding peptide sequences, the predominant of which was the disulfide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesized peptide corresponding to this sequence was found to be a competitive inhibitor of murine uPA, inhibiting its activity towards a low-molecular-mass chromogenic substrate as well as towards its natural substrate plasminogen. The K i value for inhibition as well as the K D value for binding were approx. 400 nM. Among a variety of other murine and human serine proteases, including trypsin, mupain-1 was found to be highly selective for murine uPA and did not even measurably inhibit human uPA. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified Arg 6 of mupain-1 as the P1 residue and indicated an extended binding interaction including the P5, P3, P2, P1 and P1′ residues of mupain-1 and the specificity pocket, the catalytic triad and amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging His 99 of human uPA by a tyrosine residue, the corresponding residue in murine uPA, conferred mupain-1 susceptibility on to the latter. Peptide-derived inhibitors, such as mupain-1, may provide novel mechanistic information about enzyme–inhibitor interactions, provide alternative methodologies for designing effective protease inhibitors, and be used for target validation in murine model systems.