Matrix metalloproteinases-2 and -9 (MMP-2/9) are critically involved in degradation of extracellular matrix, and their inhibition is discussed as a promising strategy against hepatic ischemia-reperfusion (I/R) injury. Here, we analyzed the role of MMP-2 and -9 for leukocyte migration and tissue injury in sham-operated mice and in mice after I/R, treated with a MMP-2/9 inhibitor or vehicle. Using zymography, we show that the MMP-2/9 inhibitor abolished I/R-induced MMP-9 activation, whereas MMP-2 activity was not detectable in all groups. As demonstrated by intravital microscopy, MMP-9 inhibition attenuated postischemic rolling and adherence of total leukocytes in hepatic postsinusoidal venules, CD4+ T cell accumulation in sinusoids, and neutrophil transmigration. These effects were associated with reduction of plasma tumor necrosis factor alpha (TNF-alpha) levels and endothelial expression of CD62P. Motility of interstitially migrating leukocytes was assessed by near-infrared reflected light oblique transillumination microscopy in the postischemic cremaster muscle. Upon MMP-9 blockade, leukocyte migration velocity and curve-line and straight-line migration distances were reduced significantly as compared with the vehicle-treated I/R group. Postischemic sinusoidal perfusion failure, hepatocellular apoptosis, and alanine aminotransferase activity were only slightly reduced after MMP-9 inhibition, whereas aspartate aminotransferase activity and mortality were significantly lower. In conclusion, MMP-9 is involved in the early recruitment cascades of neutrophils and CD4+ T cells, promotes neutrophil and T cell transmigration during hepatic I/R, and is required for motility of interstitially migrating leukocytes. MMP-9 blockade is associated with an attenuation of TNF-alpha release and endothelial CD62P expression, weakly protects from early microvascular/hepatocellular I/R damage, but improves postischemic survival.