Myocardial ischaemia/reperfusion (I/R) injury is a major cause of morbidity and mortality associated with coronary heart disease. During I/R injury, cardiomyocytes undergo cell death including apoptosis and necrosis with increased frequency, and this leads to compensatory cardiac hypertrophy, dysfunction, ventricular remodelling, and ultimately, heart failure. Accumulating evidence indicates that substance P is cardio-protective following I/R primarily due to its potent coronary vasodilator actions. However, its direct effect on cardiomyocytes in vitro remains controversial. An hypoxia/reoxygenation (H/R)-induced cell death model was established to mimic I/R injury, in which the effect of substance P (SP) pretreatment on H9C2 cardiomyocytes and the mechanism of action were explored. Substance P was demonstrated to inhibit H/R-induced apoptosis. Phosphorylated-Akt (p-Akt) was decreased in H/R groups and was increased by SP pretreatment. Inhibition of p-Akt reduced the beneficial effects of SP in reducing apoptosis, whereas activation of Akt failed to provide additional improvement in the presence of SP. This suggests a key role for Akt in the process of reduced apoptosis by SP following H/R injury. In addition, the NK1-receptor antagonist prohibited the anti-apoptotic effect of SP. This study indicates that SP pretreatment attenuates H/R-induced apoptosis via the Akt signalling pathway and the NK1-receptor.