Background: The growing number of patients awaiting heart transplantation, coupled with the worldwide donor shortage, has led to increased use of marginal organs, specifically hearts from older donors. This study compared the protective effects of two widely used preservation solutions, histidine-tryptophan-ketoglutarate (HTK) and Celsior (CEL; Sangstat Medical, Menlo Park, CA), for ischemia-reperfusion injury using a rat heterotopic heart transplantation model with older donors. Methods The hearts were excised from 16- and 80-week-old Lewis donor rats, stored in HTK, CEL, or saline for 6 hours and heterotopically transplanted into syngenic young Lewis recipients. Serum troponin I and creatine phosphokinase, graft infiltrating cells, graft apoptosis, graft proinflammatory messenger ribonucleic acid levels, and adenosine monophosphate-activated protein kinase phosphorylation were analyzed 3, 6, and 12 hours after reperfusion as markers of graft injury. Tissue adenosine triphosphate levels were measured after cold storage for 0, 6, 12, and 18 hours. Results The HTK and CEL reduced injury comparably in grafts from young donors. The recipients of grafts from older donors and stored in HTK for 6 hours had lower levels of serum troponin I and creatine phosphokinase, less upregulation of the messenger ribonucleic acid for interleukin-6, intercellular adhesion molecule-1, and tumor necrosis factor-α, fewer infiltrating cells, less apoptosis, and less phosphorylated adenosine monophosphate-activated protein kinase than recipients of grafts stored in CEL. Adenosine triphosphate levels in the hearts stored in HTK were significantly higher than those stored in CEL or saline. Conclusions Cold storage in HTK exhibited superior protective effects against ischemia-reperfusion injury of hearts from older donors in this rat transplantation model.