We have previously shown that islet emboli in the portal vein block blood flow and induce local inflammatory reaction, resulting in functional loss of islet grafts following intraportal transplantation. This study was designed to test whether Toll-like receptor (TLR) activation mediates early islet graft failure. Syngeneic islet grafts were transplanted into chemically induced diabetic mice, and TLR deficient mice were used as donors and/or recipients of islet grafts. Islet viability, proinflammatory cytokines, high-mobility group box-1 (HMGB1) and NF-kappaB activation were analyzed by bioluminesce imaging (BLI), quantitative RT-PCR (qRT-PCR) and histology. Early islet graft failure was observed in mice with intraportal islet engrafts with increased proinflammatory cytokines, HMGB1 expression, NF-kappaB activation, caspase-3 and TUNEL positive cells. Deficiency of TLR4 in donor, but not in recipient, inhibited NF-kappaB activation, reduced proinflammatory cytokines and improved viability of islet grafts. Blockade of HMGB1 with anti-HMGB1 monoclonal antibody (mAb, 2g7) inhibited inflammatory reactions, as evidenced by reduced TNFalpha and IL-1ss production, and improved islet viability. We conclude that TLR4 activation mediates early graft failure following intraportal islet transplantation. Inhibition of TLR4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation.