Leukocyte transmigration across endothelial and extracellular matrix protein barriers is dependent on adhesion and focal matrix degradation events. In the present study we investigated the role of metalloproteinase-9 (MMP-9/gelatinase B) in liver ischemia/reperfusion (I/R) injury using MMP-9-deficient (MMP-9−/−) animals and mice treated with a specific anti-MMP-9 neutralizing antibody or with a broad gelatinase inhibitor for both MMP-9 and metalloproteinase-2 (MMP-2/gelatinase A). Compared to wild-type mice, MMP-9−/− mice and mice treated with an anti-MMP-9 antibody showed significantly reduced liver damage. In contrast, mice treated with a broad gelatinase inhibitor showed rather inferior protection against I/R injury and were characterized by persistent ongoing liver inflammation, suggesting that MMP-2 and MMP-9 may have distinct roles in this type of injury. MMP-9 was mostly detected in Ly-6G and macrophage antigen–1 leukocytes adherent to the vessel walls and infiltrating the damaged livers of wild-type mice after liver I/R injury. Leukocyte traffic and cytokine expression were markedly impaired in livers of MMP-9−/− animals and in livers of mice treated with anti-MMP-9 antibody after I/R injury; however, initiation of the endothelial adhesion cascades was similar in both MMP-9−/− and control livers. We also showed that MMP-9-specific inhibition disrupted neutrophil migration across fibronectin in transwell filters and depressed myeloperoxidase (MPO) activation in vitro. Conclusion: These results support critical functions for MMP-9 in leukocyte recruitment and activation leading to liver damage. Moreover, they provide the rationale for identifying inhibitors to specifically target MMP-9 in vivo as a potential therapeutic approach in liver I/R injury. (HEPATOLOGY 2007.)