Ischemia-reperfusion injury (IRI) is an intrinsic risk of solid organ transplantation due to the cellular damage and subsequent immune activation induced by current allograft procurement protocols. Increased severity of IRI contributes to poor clinical outcomes, including early allograft dysfunction and alloimmune-mediated graft rejection, following orthotopic liver transplantation (OLT). Circulating recipient monocytes are one of the first cell populations to infiltrate the donor liver. Once activated, these monocytes can differentiate into macrophages capable of crosstalk with the adaptive immune cells responsible for the development of alloimmunity. The intracellular protein high mobility group box 1 (HMGB1) is released during IRI in mouse models and correlates with degree of IRI. HMGB1 is unique in that each of its three oxidative forms have different functions and it can bind multiple immune receptors on monocytes and macrophages, including toll-like receptor 4 (TLR4) and TLR9. Blood from IRI+ human OLT recipients activates TLR4 and TLR9. We therefore investigated whether the pro-inflammatory oxidative form of HMGB1 (disulfide HMGB1, diS-HMGB1) is increased in IRI+ patients and, if so, the mechanisms behind diS-HMGB1-induced innate and adaptive immune activation during and following OLT. First, we determined that diS-HMGB1 was increased in post-reperfusion portal vein blood of IRI+ patients and that this same blood sample induced a pro-inflammatory response in healthy volunteer monocytes in vitro. The augmented diS-HMGB1 levels in IRI+ patient blood were mediated by secretion of diS-HMGB1 from pro-inflammatory liver resident macrophages in the donor allograft that became activated during the transplant process. We confirmed the detrimental effect of increased diS-HMGB1 levels in a murine model of IRI. Second, we developed an in vitro model of human primary monocyte-to-macrophage differentiation to elucidate the specific effects of diS-HMGB1 on this process. We determined that diS-HMGB1 functions by inducing secreted factors, surface markers, and oxidative functions that allow monocytes and macrophages to create a local pro-inflammatory environment and to induce a pro-inflammatory adaptive immune response through interactions with CD4+ T cells. Finally, we report how both TLR4 and TLR9 contribute to the manifestation of this phenotype. In sum, this work provides insight into the impact of diS-HMGB1 on occurrence and severity of IRI during OLT and how the release of diS-HMGB1 at the time of transplant can have lasting impacts on the immune landscape, and therefore graft and recipient health and survival, following OLT.