There is increasing proof that organ preservation by machine perfusion is able to limit ischemia/reperfusion injury in kidney transplantation. This study was designed to compare the efficiency in hypothermic organ preservation by machine perfusion or cold storage in an animal model of kidney autotransplantation. Twelve pigs underwent left nephrectomy after warm ischemic time; the organs were preserved in machine perfusion (n = 6) or cold storage (n = 6) and then autotransplanted with immediate contralateral nephrectomy. The following parameters were compared between the two groups of animals: hematological and urine indexes of renal function, blood/gas analysis values, histological features, tissue adenosine-5'-triphosphate (ATP) content, perforin gene expression in kidney biopsies, and organ weight changes were compared before and after preservation. The amount of cellular ATP was significantly higher in organs preserved by machine perfusion; moreover, the study of apoptosis induction revealed an enhanced perforin expression in the kidneys, which underwent simple hypothermic preservation compared to the machine-preserved ones. Organ weight was significantly decreased after cold storage, but it remained quite stable for machine-perfused kidneys. The present model seems to suggest that organ preservation by hypothermic machine perfusion is able to better control cellular impairment in comparison with cold storage.