XR5118 [(3Z,6Z)-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P1 residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central β-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of α-helix F, situated above β-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to β-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.