Tumor necrosis factor-alpha (TNF-alpha), caspase-8, and complement component 5a receptor (C5aR) are known to play a crucial role in the myocardial ischemia/reperfusion (I/R) in-jury in cardiac transplantation. We hypothesized that the intracoronary infusion of TNF-alpha, caspase-8, and C5aR small interfering RNAs (siRNA) would protect cardiac allograft func-tion and improve graft survival from I/R injury-induced organ failure. I/R injury of cardiac allo-graft was induced by syngeneic rat cardiac transplantation, in which the transplanted hearts were infused with saline or different amounts of siRNA cocktail solution targeting TNF-alpha, caspase-8, and C5aR via coronary arteries, and subsequently subjected to 18 h of preser-vation at 4 degrees C in histidine-tryptophan-ketoglutarate (HTK) solution. The effects of siRNA cocktail solution on prolonged cold I/R injury were determined by assessing graft survival, histopathological changes, myeloperoxidase (MPO) activity, and malondialdehyde (MDA) concentration. The perfused siRNA cocktail solution successfully knocked down the expres-sion of TNF-alpha, caspase-8, and C5aR in vitro and in vivo. Approximately 91.7% of control hearts that underwent 18 h of cold ischemia ceased their function after transplantation; how-ever, 87.5% of cardiac allografts from the highest dose siRNA cocktail solution-pretreated hearts survived >14 days and exhibited minimal histological changes, with minimal cellu-lar infiltration, interstitial edema, and inflammation and maximal reduced MPO activity and MDA concentration in the cardiac allograft. We demonstrated the feasibility and efficiency of infusion of TNF-alpha, caspase-8, and C5aR siRNA via the intracoronary route as a promising strategy for gene silencing against I/R injury in cardiac transplantation.