Exendin‐4, a glucagon‐like peptide‐1 receptor agonist, was shown to protect against cardiac ischaemia/reperfusion (I/R) injury by suppressing oxidative stress. p66Shc, a pro‐oxidant and an apoptotic protein, is activated in the infarcted left ventricles (LVs) after induction of I/R. This study investigated if the cardiac protective effect of Exendin‐4 against I/R injury in rats involves inhibition of p66Shc and to determine the underlying mechanisms behind this. Adult male rats (n = 12/group) were divided into four groups as a sham, a sham + Exendin‐4, an I/R, and an I/R + Exendin‐4. Exendin‐4 was administered to rats 7 days before the induction of I/R. Ischaemia was induced by ligating the left anterior descending (LAD) coronary artery for 40 minutes followed by reperfusion for 10 minutes. The infarct myocardium was used for further analysis. Exendin‐4 significantly reduced infarct area (by 62%), preserved LV function and lowered serum levels of LDH and CK‐MB in I/R‐induced rats. Also, it significantly reduced LV levels of ROS and MDA and protein levels of cytochrome‐c and cleaved caspase‐3 but significantly increased levels of glutathione (GSH) and manganese superoxide dismutase (MnSOD) in LVs of I/R rats indicating antioxidant and anti‐apoptotic effects. Furthermore, it inhibited JNK and p66Shc activation and downregulated protein levels of p66Shc and NADPH oxidase with no effect on protein levels/activity of p53 and PKCβII. Of note, Exendin‐4 also increased GSH and MnSOD in LVs of control rats. In conclusion, Exendin‐4 cardioprotective effect in I/R hearts is mediated mainly by antioxidant effect and inhibition of JNK/P66Shc/NADPH oxidase.