Aliskiren, a direct renin inhibitor, has shown potent ability to attenuate hypertension. Our previous research has found that aliskiren protected against myocardial ischaemia‐reperfusion (I/R) injury and enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) in spontaneously hypertensive rats. However, whether the cardioprotective effect of aliskiren against myocardial I/R injury was eNOS‐dependent is unknown. In the present study, 12‐week‐old male eNOS knockout (eNOS−/−) and wild‐type C57BL/6J mice (WT) were orally administrated with the dose of 50 mg/kg per day of aliskiren. After a 4‐week treatment, aliskiren decreased blood pressure in eNOS−/− mice, and reduced renin‐angiotension II levels in both eNOS−/− and WT mice. Aliskiren also improved left ventricular ejection fraction (EF) and fractional shortening (FS), decreased myocardial infarct size, reduced creatine kinase (CK) and lactate dehydrogenase (LDH) activity in plasma, attenuated dihydroethidium (DHE) fluorescence and levels of malondialdehyde (MDA), enhanced superoxide dismutase (SOD) activity and total antioxidant capacity (T‐AOC) in myocardium, increased SOD and thioredoxin (Trx) proteins expression in WT mice subjected to 30 minutes of ischaemia followed by reperfusion for 24 hours. However, aliskiren failed to restore all of the above indices in eNOS−/− mice subjected to the same I/R injury. Our study indicated that aliskiren protected against myocardial I/R injury via an eNOS dependent manner.