The liver architecture plays an important role in maintaining hemodynamic balance, but the mechanisms that underlie this role are not fully understood. Hepsin, a type II transmembrane serine protease, is predominantly expressed in the liver, but has no known physiological functions. Here, we report that hemodynamic balance in the liver is regulated through hepsin. Deletion of hepsin (hepsin−/−) in mice resulted in enlarged hepatocytes and narrowed liver sinusoids. Using fluorescent microbeads and antihepsin treatment, we demonstrated that metastatic cancer cells preferentially colonized the hepsin−/− mouse liver as a result of the retention of tumor cells because of narrower sinusoids. The enlarged hepatocytes expressed increased levels of connexin, which resulted from defective prohepatocyte growth factor (pro-HGF) processing and decreased c-Met phosphorylation in the livers of hepsin−/− mice. Treatment of hepsin−/− mice with recombinant HGF rescued these phenotypes, and treatment of wild-type mice with an HGF antagonist recapitulated the phenotypes observed in hepsin−/− mice. Conclusion: Our findings show that the maintenance of hepatic structural homeostasis occurs through HGF/c-Met/connexin signaling by hepsin, and hepsin-mediated changes in liver architecture significantly enhance tumor metastasis to the liver. (HEPATOLOGY 2012;56:1913–1923)