Survival of transplanted hearts is often limited by cold ischemia time. Here, we assessed the effects of the small molecular compound TJ-M2010-5 on graft preservation. In a cardiac cold ischemia/reperfusion model, TJ-M2010-5 ameliorated myocardial ischemia/reperfusion injury (MIRI) in histidine-tryptophan-ketoglutarate (HTK) organ preservation solution. When applied in HTK solution and on donors/recipients respectively, TJ-M2010-5 exerted optimal effects when applied as an additive in the HTK solution. TJ-M2010-5-administered mice exhibited shorter rebeating time; higher beating score; stronger and more regular sinus heart rate; and amelioration of apoptosis, inflammatory reactions, and myocardial injury. Mechanistically, TJ-M2010-5 inhibited the expression of key molecules in the toll-like receptor (TLR) signaling pathway and affected downstream proteins by inhibiting myeloid differentiation factor 88 homodimerization, thereby decreasing myocardial injury. Thus, TJ-M2010-5 may exert protective effects against MIRI by blocking the TLR signaling pathway. Our findings may lead to novel approaches for organ preservation, thereby reducing organ abandonment and improving recipient prognosis. The role of the TLR signaling pathway in MIRI progress and operation procedure of the MIRI model in vivo are presented in a graphical abstract (Online Abstract Figure).