Venous thromboembolism (VTE), which includes pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major cause of morbidity. Vascular endothelial cell injury, venous stasis, and/or alterations in blood hypercoagulability can propagate VTE. DVT is characterized by thrombi that form on the surface of activated endothelium in both the vein valve pockets and dilated sinuses of the lower limbs. Current clinical anticoagulant therapies prevent further propagation of venous thrombi but do not promote thrombus resolution and do not modulate the underlying inflammatory processes that promote chronic post-thrombotic syndrome (PTS). The serine protease urokinase-plasminogen activator (uPA) is a crucial mediator of fibrinolysis during thrombus resolution. We have shown, using a clinically relevant mouse model of DVT, that removal of the natural inhibitors of uPA, plasminogen activator inhibitor (PAI) type -1 (PAI-1) and type-2 (PAI-2), leads to accelerated thrombus resolution in vivo. Furthermore, we found that PAI-2-deficient thrombi have accelerated fibrinolysis as measured through fibrin degradation product (FDP) generation in a manner like that of PAI-1 deficiency. PAI-2 is widely known to be produced by macrophages, however, we find uPA-dependent macrophage fibrinolysis is mediated by PAI-1 and not PAI-2 expression. Interestingly, PAI-2 deficiency impacts early accumulation of fibrinolytic neutrophils, enhancing CXCL2-mediated neutrophil migration into the thrombus, possibly altering the inflammatory environment. Taken together, our novel findings have identified a potential mechanism by which PAI-2 and PAI-1 impede venous thrombus resolution and thereby have the potential to lead to new therapies to improve outcomes for patients with VTE.