This study aims to explore the protective effects of nobiletin against hepatic ischemia-reperfusion (IR) injury after liver transplantation. Kupffer cells (KCs) were activated and co-cultured with different concentration of nobiletin for 24h in vitro, inflammatory products and activity of TLR4/NF-κB signaling pathway were detected. Sprague-Dawley rats were selected and underwent orthotopic liver transplantation. Donors were injected intravenously with nobiletin (50mg/kg) or saline solution, once a day for 1 week before the surgery. Recipients were randomly paired and sacrificed at the indicated time points (3, 6, and 24h after the surgery), the graft liver tissues and blood samples were collected for analysis. Hepatic function, inflammatory mediators, apoptosis of hepatocytes, histological changes, KCs and CD4+ T-lymphocyte infiltration were assessed. Results showed nobiletin dose-dependently suppressed the expression of inflammatory mediators and the activity of TLR4/NF-κB signaling pathway in activated KCs. Furthermore, nobiletin alleviated liver damage induced by IR in vivo, significantly decreased the serum levels of alanine aminotransferase, aspartate transaminase, inflammatory cytokines and alleviated the histopathology changes. Moreover, liver in the nobiletin treated group exhibited less KCs and CD4+ lymphocyte infiltration and lower hepatocyte apoptosis after operation. In addition, activity of TLR4/NF-κB signaling pathway in KCs was also suppressed, consistent with the results in vitro. Collectively, Nobiletin can ameliorate IR injury after liver transplantation and may be a promising new strategy to protect against liver IR injury.