Previously, we showed that bikunin, a Kunitz-type protease inhibitor, inhibits invasion and metastasis in several types of cancer cells possibly through suppression of upregulation of urokinase-type plasminogen activator (uPA) expression. Bikunin corresponds to a light chain of the inter-alpha inhibitor. To explore critical role of endogenous bikunin, we used bikunin knockout (Bik−/−) mice. Here, we show that 1) higher frequency of spontaneous 3LL lung metastasis was observed in Bik−/− mice compared to Bik+/+ mice, suggesting that bikunin deficiency increases the sensitivity of mice to lung metastasis; 2) administration of exogenous bikunin caused a significant reduction of lung metastasis in Bik−/− and Bik+/+ mice; 3) primary and metastatic tumors significantly upregulated uPA and PAI-1 expression in Bik−/− mice relative to Bik+/+ mice at least through phosphorylation of ERK1/2 and 4) exogenous bikunin suppressed phosphorylation of ERK1/2 and upregulation of uPA and PAI-1 expression in 3LL cells in response to G-CSF. These data allow us to conclude that the increased sensitivity of Bik−/− mice to lung metastasis in vivo is due to a lack of circulating proteins of the inter-alpha inhibitor family, especially bikunin. © 2005 Wiley-Liss, Inc.