We have previously reported that the serpin plasminogen activator inhibitor-1 activates the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signalling pathway and stimulates cell migration by binding to the low-density lipoprotein receptor-related protein. All the free forms (cleaved, latent or active) of this inhibitor were shown to be motogenic. However, the plasminogen activator inhibitor-1 can also interact with vitronectin which acts as a cofactor by increasing the half-life of the active form of the serpin. Since vitronectin influences most of the biological functions of the plasminogen activator inhibitor-1, we explored the effects of vitronectin on signalling and cell migration induced by this serpin. We found that the interaction between vitronectin and the plasminogen activator inhibitor-1 suppressed signalling and cell migration. In fact, a purified vitronectin(1-97)/plasminogen activator inhibitor-1 complex was not chemotactic. Vitronectin interaction with the plasminogen activator inhibitor-1 blocks the binding of this serpin to its motogenic receptor, the low-density lipoprotein receptor-related protein. Consequently, vitronectin inhibits the activation of the Janus kinase/signal transducer and activator of transcription signalling pathway by the plasminogen activator inhibitor-1 and subsequent cell migration. In conclusion, we have unveiled a new inhibitory role of vitronectin, which turns off the intracellular signalling and migration-promoting activity of the plasminogen activator inhibitor-1. Thus, the motogenic (cleaved, latent or active) and non-motogenic (in complex with vitronectin) forms of the plasminogen activator inhibitor-1 have different properties that may explain the rather contrasting physiological and pathological roles of this serpin.