Proteinaceous protease inhibitors can strongly and specifically inhibit cognate proteases, but their use as pharmaceuticals is limited by their size. As such, the development of effective protease peptide inhibitors would be beneficial for biochemical studies and drug discovery. In this study, we applied a phage display system to select subtilisin BPN′‐binding peptides and evaluated their inhibitory activities against subtilisin BPN′. A 12mer peptide with an intramolecular disulfide bond inhibited subtilisin BPN′ (Ki value of 13.0 nm). Further mutational analyses of the peptide resulted in the development of a short peptide inhibitor against subtilisin BPN′ that showed high inhibitory activity and binding affinity (Ki value of 0.30 nm). This activity was found to be derived from the conformational rigidity caused by the intramolecular disulfide bond and the small residue at the P1′ site and from the interaction of the P4 and P6′ residues with subtilisin BPN′.