Malformin A₁, a cyclopentapeptide of fungal origin, enhances cellular fibrinolytic activity depending on the existence of a cofactor in blood plasma. However, the nature of this cofactor remains unknown. Here, we report that vitronectin acts as a plasma cofactor of malformin A₁. We purified the cofactor from bovine plasma by activity-based fractionation, and confirmed that vitronectin in conjunction with plasminogen supports the activity of malformin A₁ to promote the fibrinolytic activity of U937 cells. Malformin A₁ action was abolished by Arg-Gly-Asp peptide (a competitor of vitronectin-integrin binding), wortmannin (an inhibitor of signaling kinases), and cytochalasin B (an inhibitor of actin polymerization). Changes in actin organization and a decrease in filopodia were observed in cells treated with malformin A₁ and plasma. A focal localization of plasminogen on the cell surface was augmented by malformin A₁, whereas the amount of cell-surface-bound plasminogen was minimally altered by the treatment. Our results suggest the involvement of cytoskeletal reorganization via vitronectin signaling in the cellular fibrinolytic activity-enhancing action of malformin A₁.