This study investigated the protective effect of dapagliflozin on H9c2 cardiomyocyte function under high glucose and hypoxia/reoxygenation (HG-H/R) conditions and identified the underlying molecular mechanisms. Dapagliflozin reduced the level of lactate dehydrogenase and reactive oxygen species in cardiomyocytes under HG-H/R conditions and was accompanied by a decrease in caspase-3/9 activity. In addition, Dapagliflozin significantly reduced mitochondrial permeability transition pore opening and increased ATP content, accompanied by upregulation of OPA1 with autophagy-related protein molecules and activation of the AMPK/mTOR signalling pathway in HG-H/R treated cardiomyocytes. OPA1 knockdown or compound C treatment attenuated the protective effects of dapagliflozin on the cardiomyocytes under HG-H/R conditions. Downregulation of OPA1 expression increased mitochondrial intolerance in cardiomyocytes during HG-H/R injury and the AMPK-mTOR-autophagy signalling is a key mechanism for protecting mitochondrial function and reducing cardiomyocyte apoptosis. Collectively, dapagliflozin exerted protective effects on the cardiomyocytes under HG-H/R conditions. Dapagliflozin attenuated myocardial HG-H/R injury by activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy.