The N-terminal fragment of prolactin (16K PRL) inhibits tumor growth by impairing angiogenesis, but the underlying mechanisms are unknown. Here, we found that 16K PRL binds the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1), which is known to contextually promote tumor angiogenesis and growth. Loss of PAI-1 abrogated the antitumoral and antiangiogenic effects of 16K PRL. PAI-1 bound the ternary complex PAI-1-urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR), thereby exerting antiangiogenic effects. By inhibiting the antifibrinolytic activity of PAI-1, 16K PRL also protected mice against thromboembolism and promoted arterial clot lysis. Thus, by signaling through the PAI-1-uPA-uPAR complex, 16K PRL impairs tumor vascularization and growth and, by inhibiting the antifibrinolytic activity of PAI-1, promotes thrombolysis.