Waiting lists for transplantation have stimulated interest in the use of non-heart-beating donor (NHBD) organs. Recent studies on organ preservation have shown advantages of machine perfusion (MP) over cold storage (CS). To supply the liver with specific nutrients during MP, the preservation solution Polysol was developed. The aim of our study was to compare CS in University of Wisconsin solution (UW) with MP using UW-gluconate (UW-G) or Polysol in an NHBD model. After 30 minutes of warm ischemia, livers were harvested from rats for preservation by either CS, MP-UW-G, or MP-Polysol. After 24 hours of preservation, livers were reperfused with Krebs-Henseleit buffer (KHB). Perfusate samples were analyzed for liver damage and function. Biopsies were examined by hematoxylin and eosin staining and transmission electron microscopy. Liver damage was highest after CS compared with the MP groups. MP using Polysol compared with UW-G resulted in less aspartate aminotransferase (AST) and alanine aminotransferase (ALT) release. Perfusate flow, bile production, and ammonia clearance were highest after MP-Polysol compared with CS and MP-UW-G. Tissue edema was least after MP-Polysol compared with CS and MP-UW-G. In conclusion, preservation of the NHBD rat liver by hypothermic MP is superior to CS. Furthermore, MP using Polysol results in better-quality liver preservation compared with using UW-G.