1. Astragaloside IV (AST-IV) is purified from a natural plant product. Previous studies have shown that AST-IV has anti-oxidant activity. In the present study, we investigated the effect and mechanism of action AST-IV on rat cardiomyocytes subjected to hypoxic conditions (up to 12 h). 2. Cardiomyocytes were prepared from neonatal rats and cultured under normoxic or hypoxic conditions in the absence or presence of AST-IV (12.5, 25 or 50 microg/mL). Cell viability, malondialdehyde (MDA) levels, activity and expression of superoxide dismutase (SOD)-1 (mRNA and protein levels determined by reverse transcription-polymerase chain reaction and western blotting, respectively) and reactive oxygen species (ROS; determined by 2',7'-dichlorodihydrofluorescein diacetate) were investigated under these culture conditions. Intracellular localization of AST-IV was tested using fluorescein isothiocyanate-labelled AST-IV. 3. Hypoxic culture reduced the viability of cardiomyocytes, which was improved following treatment with 25 or 50 microg/mL AST-IV. Under hypoxic conditions, MDA levels were double those under control conditions. Astragaloside IV (25 and 50 microg/mL) dose-dependently reduced the increase in MDA seen in hypoxic cardiomyocytes. 4. Fluorescein isothiocyanate-labelled AST-IV entered cardiomyocytes and was localized mainly within the cytoplasm. 5. Under hypoxic conditions, SOD-1 activity was decreased, but mRNA and protein expression increased, compared with normoxia. Following treatment with 25 microg/mL AST-IV, SOD-1 activity and expression were increased under both normoxic and hypoxic conditions. The ROS scavenging effect of AST-IV was abolished in the presence of the SOD inhibitor sodium diethyl dithiocarbamate (25 micromol/L). 6. These in vitro results show that AST-IV protects cardiomyocytes from oxidative stress-mediated injury under hypoxic conditions. A major part of this action is achieved by upregulation of SOD-1 content and activity within the cell cytoplasm.