Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS-D chloroacetate esterase stains, and immunohistochemistry (anti–Ly-6G). The expression of pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2, and CXCL-10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and caspase-3 activity. In addition, toll-like receptor 4 (TLR4) expression was diminished in NEI-pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939–947, 2009. © 2009 AASLD.