Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is one of the major pathways for cytokine signal transduction. However, the role of the JAK/STAT pathway in liver ischemia/reperfusion is not clear. This study focuses on Janus kinase-2 (JAK2), which functions upstream of signal transducer and activator of transcription 1 (STAT1) in JAK/STAT, and its role in the mechanism of liver ischemia/reperfusion injury (IRI). Partial warm ischemia was produced in the hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 hours of reperfusion. Mice were treated with a JAK2 inhibitor (tyrphostin AG490; 40 mg/kg intraperitoneally) or vehicle 60 minutes prior to ischemic insult. JAK2 blockade resulted in a significant reduction of hepatocyte apoptosis and liver injury. Macrophage and neutrophil infiltration, as assessed by immunohistochemistry, was markedly decreased in AG490-treated livers in comparison with controls. The expression of pro-inflammatory cytokines [tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1b] and chemokines [chemokine (C-X-C motif) ligand 10 (CXCL-10) and CXCL-2] was also significantly reduced in the AG490-treated group in comparison with controls. AG490-treated livers showed fewer cells positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and reduced cleaved caspase-3 protein expression in parallel with increased Bcell lymphoma extra large expression. We employed AG490 (75 mM) in primary bone marrow-derived macrophage (BMM) and hepatoma cell (CRL1830) cultures, which were both stimulated with lipopolysaccharide (LPS; 10 ng/mL). In BMM cultures, AG490 depressed otherwise LPS-induced pro-inflammatory gene expression programs (IL-6, IL-12p40, IL-1b, CXCL-10, and inducible nitric oxide synthase). In hepatoma cells, AG490 reduced cleaved caspase-3 expression. Moreover, JAK2 blockade inhibited STAT1 and STAT3 phosphorylation. This is the first report documenting that JAK2 signaling is essential in the pathophysiology of liver IRI, as its selective blockage ameliorated the disease process and protected livers from inflammation and apoptosis.