Carbon monoxide (CO) exposure of an islet donor frequently leads to islet allograft long-term survival and tolerance in recipients. We show here that CO confers its protective effects at least in part by suppressing Toll-like receptor 4 (TLR4) up-regulation in pancreatic cells. TLR4 is normally up-regulated in islets during the isolation procedure; donor treatment with CO suppresses TLR4 expression in isolated islets as well as in transplanted grafts. TLR4 up-regulation allows initiation of inflammation, which leads to islet allograft rejection; islet grafts from TLR4-deficient mice survive indefinitely in BALB/c recipients and show significantly less inflammation at various days after transplantation compared with grafts from a control donor. Isolated islets preinfected with a TLR4 dominant negative virus before transplantation demonstrated prolonged survival in recipients. Despite the salutary effects of TLR4 suppression, HO-1 expression is still needed in the recipient for islet survival: TLR4-deficient islets were rejected promptly after being transplanted into recipients in which HO-1 activity was blocked. In addition, incubation of an insulinoma cell line, TC3, with an anti-TLR4 antibody protects those cells from cytokine-induced apoptosis. Our data suggest that TLR4 induction in cells is involved in cell death and graft rejection after transplantation. CO exposure protects islets from rejection by blocking TLR4 up-regulation.--Goldberg, A., Parolini, M., Chin, B. Y., Czismadia, E., Otterbein, L. E., Bach, F. H., Wang, H. Toll-like receptor 4 suppression leads to islet allograft survival.