Introduction: The urokinase-type Plasminogen Activator (uPA) catalyzes the rate-limiting step in the fibrinolysis pathway. How the activity of uPA is regulated is not well characterized and its understanding could unravel alternative ways of tuning fibrinolysis. The uPA has three domains: an EGF-like and a Kringle domain linked through a disordered linker to a serine protease domain. The linker binds to the protease domain through a disulfide bond that is located 22.7 Å away from the catalytic triad. There is no crystal structure available for full-length uPA and it is unknown if these domains interact among themselves. We recently showed that full-length uPA is more active than the protease domain by itself. Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) revealed that the protease domain was less dynamic in the presence of the EGF and Kringle domains, however, our data did not show any interface between the domains. We hypothesize that the linker is allosterically regulating the protease domain. Methods: We mutated two residues in the linker of full-length uPA (S138D and P141A) and measured their activity using a small substrate analog and the endogenous substrate Plasminogen. In addition, changes in dynamics of the protease domain were evaluated using HDXMS. Results: The S138D mutation decreased the activity of the enzyme towards both substrates, while the P141A mutation increased the activity towards the small chromogenic substrate and did not alter the activity towards Plasminogen. HDXMS revealed how the dynamics of different loops in the protease domain were affected by the mutations. The 220s loop was less dynamic in the P141A mutant, however the 110s loop, the activation loop and the β9 strand were more dynamic on this mutant compared to the wildtype. The 110s, 170s and 140s loops together with the activation loop and the β9 strand were more dynamic in the S138D mutant. Interestingly, all these loops are between 20 to 40 Å away from the cysteine that connects the linker to the protease domain, as measured from PDB: 1C5W. Conclusions: Our results support the hypothesis that the long-range dynamic allostery in full-length uPA is mediated by the disordered linker. In addition, this allostery regulates the activity and dynamics of the protease domain of uPA.