Hypoxia‐inducible factor 1α (HIF‐1α) plays a critical role in the apoptotic process during cardiac ischaemia/reperfusion (I/R) injury. This study aimed to investigate whether post‐treatment with dexmedetomidine (DEX) could protect against I/R‐induced cardiac apoptosis in vivo and in vitro via regulating HIF‐1α signalling pathway. Rat myocardial I/R was induced by occluding the left anterior descending artery for 30 minutes followed by 6‐hours reperfusion, and cardiomyocyte hypoxia/reoxygenation (H/R) was induced by oxygen‐glucose deprivation for 6 hours followed by 3‐hours reoxygenation. Dexmedetomidine administration at the beginning of reperfusion or reoxygenation attenuated I/R‐induced myocardial injury or H/R‐induced cell death, alleviated mitochondrial dysfunction, reduced the number of apoptotic cardiomyocytes, inhibited the activation of HIF‐1α and modulated the expressions of apoptosis‐related proteins including BCL‐2, BAX, BNIP3, cleaved caspase‐3 and cleaved PARP. Conversely, the HIF‐1α prolyl hydroxylase‐2 inhibitor IOX2 partly blocked DEX‐mediated cardioprotection both in vivo and in vitro. Mechanistically, DEX down‐regulated HIF‐1α expression at the post‐transcriptional level and inhibited the transcriptional activation of the target gene BNIP3. Post‐treatment with DEX protects against cardiac I/R injury in vivo and H/R injury in vitro. These effects are, at least in part, mediated via the inhibition of cell apoptosis by targeting HIF‐1α signalling.