Neutrophil extravasation from sinusoids is a critical step for acute inflammatory tissue injury. However, the role of sinusoidal endothelial cells (SECs) in this process remains unclear. Matrix metalloproteinases (MMPs) have been shown to involve gap formation in SECs in several liver diseases. Therefore, the present study examined SEC modifications elicited by galactosamine (Gal)/endotoxin (ET). Treatment of male C3Heb/FeJ mice with Gal/ET or Gal/TNF caused the formation of numerous gaps in SECs at 4 hours when no neutrophil extravasation occurred. Six hours after Gal/ET or Gal/TNF treatment, blood elements started to penetrate to the extra-sinusoidal space through large gaps. Treatment with ET alone caused sinusoidal neutrophil accumulation but no gap formation, neutrophil extravasation or hemorrhage. Gal/ET treatment increased hepatic MMP-2 and MMP-9 mRNA expression (6.7-fold and 11-fold, respectively). Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, a MMP-2/MMP-9 inhibitor (5 mg/kg), minimized gap formation after Gal/ET and Gal/TNF treatment. The MMP inhibitor reduced injury only in the Gal/ET model mainly due to reduced TNF formation. The MMP inhibitor attenuated sinusoidal neutrophil accumulation at 6 hours but failed to attenuate Gal/TNF-induced liver injury at 7 hours due to excessive apoptosis. These results suggest Gal/ET or Gal/TNF activates MMPs, which are responsible for SEC gap formation. Although the initial appearance of gap formation is independent of neutrophils, the gaps allow initial contact of neutrophils with damaged hepatocytes. In addition, MMP activation promotes neutrophil accumulation in sinusoids.