Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, β2-glycoprotein I (β2GPI), stimulates t-PA–dependent plasminogen activation in the fluid phase and within a fibrin gel. The region within β2GPI responsible for stimulating t-PA activity is at least partially contained within β2GPI domain V. β2GPI bound t-PA with high affinity (Kd ~ 20 nM), stimulated t-PA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (kcat/Km) of t-PA–mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of β2GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following β2GPI repletion. Finally, stimulation of t-PA–mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies, as well as by anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS). These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS.