Abstract Background Arctic fish survive subzero temperatures by producing a family of antifreeze proteins (AFPs) that noncolligatively lower the freezing temperature of their body fluids. We report 24-hour storage of mammalian hearts for transplantation at subzero temperatures using AFPs derived from arctic fish. Methods Forty-two heterotopic transplantations were performed in isoimmune Sprague-Dawley rats. Harvested hearts were retrogradely infused with cold 4°C University of Wisconsin (UW) solution and were preserved in a specialized cooling bath at two target temperatures, 4°C and −1.3°C for 12,18, and 24 hours (6 experiments/group). Preservation solutions were UW alone for the 4°C group, and UW with 15 mg/mL AFP III for the −1.3°C group. After hypothermic storage the hearts were heterotopically transplanted into isoimmune rats. Viability was assessed and graded on a scale of 0 to 6 (0 = no contractions to 6 = excellent contractions). Transplanted hearts were then fixed in vivo and were subject to electron microscopy and histopathologic examination. Results None of the hearts preserved at −1.3°C in UW/AFP III solution froze. All control hearts preserved at −1.3°C without AFP protection froze and died at reperfusion. Viability of hearts preserved at −1.3°C in UW/AFP III solution was significantly better after 18 hours of preservation, 30 and 60 minutes after reperfusion (median, 5 versus 3 and 6 versus 3, respectively; p < 0.05) and after 24 hours of preservation 30 and 60 minutes after reperfusion (median, 4.5 versus 1.5 and 5 versus 2, respectively; p < 0.05). Histologic and electron microscopy studies demonstrated better myocyte structure and mitochondrial integrity preservation with UW/AFP III solution. Conclusions Antifreeze proteins prevent freezing in subzero cryopreservation of mammalian hearts for transplantation. Subzero preservation prolongs ischemic times and improves posttransplant viability.