Ischemia reperfusion injury (IRI) leads to significant tissue damage in liver surgery. Polyethylene glycols (PEGs) are water soluble nontoxic polymers that have proved their effectiveness against IRI. The objective of our study was to investigate the potential protective effects of intravenous administration of a high molecular weight PEG of 35 kDa (PEG 35) in steatotic livers subjected to cold ischemia reperfusion. In this study, we used isolated perfused rat liver model to assess the effects of PEG 35 intravenous administration after prolonged cold ischemia (24 h, 4°C) and after reperfusion (2 h, 37°C). Liver injury was measured by transaminases levels and mitochondrial damage was determined by confocal microscopy assessing mitochondrial polarization (after cold storage) and by measuring glutamate dehydrogenase activity (after reperfusion). Also, cell signaling pathways involved in the physiopathology of IRI were assessed by western blot technique. Our results show that intravenous administration of PEG 35 at 10 mg/kg ameliorated liver injury and protected the mitochondria. Moreover, PEG 35 administration induced a significant phosphorylation of prosurvival protein kinase B (Akt) and activation of cytoprotective factors e-NOS and AMPK. In conclusion, intravenous PEG 35 efficiently protects steatotic livers exposed to cold IRI.