INTRODUCTION: Long-term normothermic machine perfusion (LT-NMP) enables the assessment and optimization of livers for days and, potentially, weeks. However, models of LT-NMP have only been described for human and pig livers, which are resource intensive and impractical for laboratory research. Cost-effective small animal models of LT-NMP are needed for future research. This study aimed to develop a system for LT-NMP of rat livers for up to 72 h. METHODS: This study was performed in two stages: the development phase (n = 20) and validation phase (n = 5). The perfusion system included an organ reservoir, pump, heat exchanger, long-term oxygenator, and dialysis. Hormonal and nutritional support were continuously infused. During the validation phase, five consecutive grafts were perfused using our protocol. At 72 h postreperfusion, grafts were assessed for viability, which was based on hemodynamic stability, mitochondrial function, bile production, and metabolic activity. RESULTS: Rodent livers were supported up to 107 h using our LT-NMP protocol. All grafts in the validation phase remained viable at 72 h (n = 5/5). The median oxygen consumption and bile production at 72 h were 0.079 mLO CONCLUSIONS: This is the first study to report LT-NMP of rodent livers up to 5 days. Using our protocol, rat livers could reliably be supported until 72 h. This model provides a greater opportunity to investigate novel therapeutics to assess, optimize, and regenerate liver grafts for transplantation.