Isosteviol sodium (STVNa), which is a derivate of the natural sweet‑tasting glycoside stevioside, has recently been developed and it has been determined that this compound exhibits neuro‑ and cardio‑protective properties. In the current study, whether STVNa interferes with the development of cardiac hypertrophy, which is induced by isoprenaline (Iso), was investigated in an experimental rat model. Rats were treated with a vehicle (0.9% NaCl; control), isoprenaline (Iso; 5 mg/kg) or Iso (5 mg/kg) with STVNa (4 mg/kg; Iso + STVNa). Cardiomyocytes were isolated using enzymatic dissociation and were treated with 5 µM Iso for 24 h and co‑treated with 5 µM STVNa. Brain natriuretic peptide (BNP) mRNA expression was determined using PCR analysis. Cell surface area, intracellular reactive oxygen species (ROS), mitochondrial transmembrane potential (ΔΨm), cytoplasmic Ca2+ and Ca2+ and contractile function were examined using a laser scanning confocal microscope. The current study demonstrated that STVNa inhibited Iso‑induced cardiac hypertrophy by inhibiting cardiomyocyte size. STVNa significantly reduced cell surface area and decreased BNP mRNA expression in ventricular cardiomyocyte Iso‑induced hypertrophy. STVNa was also revealed to restore ΔΨm and reduce ROS generation and intracellular Ca2+ concentration when compared with the Iso‑treated group. Additionally, STVNa preserved Ca2+ transients in hypertrophic cardiomyocytes. In conclusion, the present study demonstrated that STVNa protects against Iso‑induced myocardial hypertrophy by reducing oxidative stress, restoring ΔΨm and maintaining Ca2+ homeostasis.