We previously reported that normothermic ex vivo machine perfusion (NEVKP) is superior in terms of organ protection compared to static cold storage (SCS), which is still the standard method of organ preservation, but the detailed mechanism is unclear. We used a large animal kidney autotransplant model to evaluate mitochondrial function during organ preservation and after kidney transplantation utilizing live cells extracted from renal tissue. Porcine kidneys stored under normothermic perfusion showed preserved mitochondrial function and higher ATP levels compared to kidneys stored at 4°C (SCS). Mitochondrial respiration and ATP levels were further enhanced when AP39, a mitochondria-targeted hydrogen sulfide donor, was administered during warm perfusion. Correspondingly, the combination of NEVKP and AP39 was associated with decreased oxidative stress and inflammation, and with improved graft function after transplantation. In conclusion, our findings suggest that the organ-protective effects of normothermic perfusion are mediated by maintenance of mitochondrial function and enhanced by AP39 administration. AP39, in combination with warm perfusion, has the potential to become the new standard of care for long-term renal preservation.