The therapeutic potential of cardiac μ-opioid receptors in ischaemia-reperfusion (I/R) injury during opioid-modulating diseases, such as heart failure, is unknown. We aimed to explore the changes of cardiac μ-opioid receptor expression during heart failure, and its role in opioid-induced cardioprotection. Rats received doxorubicin (DOX) or were subjected to coronary artery ligation to induce heart failure, or received normal saline (NS) as control. Hearts from NS or DOX rats were isolated and subjected to myocardial ischaemia and reperfusion in an in vitro perfusion system. The opioid D-Ala,2N-MePhe,4 Gly-ol-enkephalin (DAMGO), with a high μ-opioid receptor specificity, morphine, and remifentanil were administrated before I/R with or without opioid receptor antagonists, or an extracellular signal-regulated kinase (ERK) inhibitor. Cardiac μ-opioid receptor mRNA concentrations were 3.2 times elevated in DOX-treated rats compared with NS rats, while cardiac μ-opioid receptor protein concentrations showed 6.1- and 3.5-fold increases in DOX-treated and post-infarcted rats, respectively. DAMGO reduced I/R-caused infarct size, expressed as the ratio of area at risk, from 0.50 (0.04) to 0.25 (0.03) in failing rat hearts, but had no effect on infarct size in control hearts. DAMGO promoted phosphorylation of ERK and glycogen synthase kinase (GSK)-3β only in failing hearts. DAMGO-mediated cardioprotection was blocked by an ERK inhibitor. The μ-opioid receptor antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) prevented morphine- and remifentanil-induced cardioprotection and phosphorylation of ERK and GSK-3β in failing hearts. In contrast, δ- and κ-opioid receptor selective antagonists were less potent than CTOP in the failing hearts. Cardiac μ-opioid receptors were substantially up-regulated during heart failure, which increased DAMGO-induced cardioprotection against I/R injury.