Background: Increasing allograft ischemic time is a significant risk factor for mortality following heart transplantation (HTx). The purpose of this study was to evaluate the protective effects of histidine-tryptophan-ketoglutarate (HTK) and Celsior (CEL) using a rat HTx model with prolonged cold storage. Methods: The hearts were excised from donor rats, stored in cold preservation solution for either 6 or 18 hours, and heterotopically transplanted into syngeneic recipients. Serum creatine phosphokinase (CPK), serum troponin I, graft-infiltrating cells, graft mRNA levels for inflammatory mediators, and tissue adenosine triphosphate (ATP) levels were analyzed, as markers of graft injury. Results: The recipients of grafts stored in HTK for 18 hours of prolonged cold ischemia had lower levels of serum CPK and tissue malondialdehyde, less upregulation of the mRNAs for IL-6 and inducible nitric oxide synthase, less apoptosis, and higher ATP levels than those receiving grafts stored in CEL and Saline. Cardiac contraction 3 hours after reperfusion was observed in 43% of the cardiac grafts stored in HTK for 18 hours, while no cardiac wall movement was seen in grafts stored in either saline or CEL. Conclusion: Cold storage in HTK exhibited superior protective effects against prolonged cold ischemia in a syngeneic rat transplantation model.