Ischemia-reperfusion injury (IRI), an innate immunity-driven local inflammation, remains the major problem in clinical organ transplantation. T cell immunoglobulin and mucin domain (TIM-3) – Galectin-9 (Gal-9) signaling regulates CD4+ Th1 immune responses. Here, we explored TIM-3 – Gal-9 function in a clinically relevant murine model of hepatic cold storage and orthotopic liver transplantation (OLT). C57BL/6 livers, preserved for 20h at 4°C in UW solution, were transplanted to syngeneic mouse recipients. Up-regulation of TIM-3 on OLT-infiltrating activated CD4+ T cells was observed in the early IRI phase (1h). By 6h of reperfusion, OLTs in recipients treated with a blocking anti-TIM-3 Ab were characterized by: 1/ enhanced hepatocellular damage (sALT levels, liver Suzuki's histological score); 2/ polarized cell infiltrate towards Th1/Th17-type phenotype; 3/ depressed T cell exhaustion markers (PD-1, LAG3); and 4/ elevated neutrophil and macrophage infiltration/activation. In parallel studies, adoptive transfer of CD4+ T cells from naïve WT, but not from TIM-3 Tg donors, readily recreated OLT damage in otherwise IR-resistant RAG−/− test recipients. Furthermore, pre-treatment of mice with rGal-9 promoted hepatoprotection against preservation-association liver damage, accompanied by enhanced TIM-3 expression in OLTs. Thus, CD4+ T cell-dependent “negative” TIM-3 costimulation is essential for hepatic homeostasis and resistance against IR stress in OLTs.