Bruton's tyrosine kinase (Btk) is a central player in multiple signaling pathways of lymphoid and myeloid cells. Myeloid cells are crucial early effectors in organ ischemia/reperfusion injury (IRI). BTKB66 is a selective, irreversible inhibitor of Btk. In this study, we hypothesized that Btk inhibition would reduce hepatocellular injury in a murine model of liver warm hepatic ischemia and reperfusion.First, BTKB66 was tested in in vitro models of LPS-mediated neutrophil and macrophage activation. Then, to assess its efficacy in vivo, BTKB66 was administered orally to mice for seven days prior to subjecting them to 90 minutes of warm hepatic ischemia followed by reperfusion for 6 or 24 hours. Clinical and pathologic features in the livers, including AST, ALT, and a panel of cytokines and chemokines, were examined.BTKB66 potently inhibited LPS-mediated activation of bone-marrow derived neutrophils and macrophages in vitro. It also reduced the severity of IRI as determined by AST and ALT levels, as well as immune cell infiltrates. BTKB66 significantly decreased hepatic markers of sterile inflammation, such as CXCL1, CXCL2, and CXCL10, in parallel with depression of serum markers of the myeloid cell activation, such as CCL5, CCL11, and CXCL5.BTKB66 treatment ameliorated hepatocellular injury in a well-established model of liver partial warm ischemia and in situ reperfusion. These findings confirm that neutrophil recruitment and activation play an essential role in IR-stress, and that targeting Btk activity may provide a useful approach for preventing hepatocellular damage and improving outcomes in liver transplantation.