Matrine has been found to affect cell viability and function. In the present study, we explored the cardioprotective role of matrine in cardiomyocyte damage under hypoxia/reoxygenation. In vitro, cardiomyocyte hypoxia/reoxygenation was used to mimic ischemia/reperfusion injury in the presence of matrine. After exposure to hypoxia/reoxygenation, cardiomyocyte viability was reduced and cell apoptosis was increased; this alteration was inhibited by matrine. At the molecular levels, Sirt3 and AMPK were significantly downregulated by hypoxia/reoxygenation injury whereas matrine administration was able to upregulate Sirt3 and AMPK expression and activity in the presence of hypoxia/reoxygenation. Interestingly, inhibition of Sirt3/AMPK pathway abolished the cardioprotective action of matrine on cardiomyocyte in the presence of hypoxia/reoxygenation injury, resulting into cardiomyocyte viability reduction and cell death augmentation. Altogether, our results demonstrated a novel role played by matrine in regulating cardiomyocyte viability and death in the presence of hypoxia/reoxygenation, with a potential application in the clinical practice for the treatment of patients with myocardial infarction. © 2020 Informa UK Limited, trading as Taylor & Francis Group.