Aim: To examine the antihypertrophic effect of ginsenoside Rb1 (Rb1) induced by prostaglandin F2α (PGF2α) in vitro and to investigate the possible mechanisms involved in the calcineurin (CAN) signal transduction pathway. Methods: The cardiomyocyte hypertrophy induced by PGF2α and the antihypertrophic effect of Rb1 were evaluated in primary culture by measuring the cell diameter, protein content, and atrial natriuretic peptide (ANP) mRNA expression. ANP and CaN mRNA expressions, CaN and its downstream effectors NFAT3 and GATA4 protein expressions, and the intracellular free Ca2+ concentration ([Ca2+]i) were assayed by RT-PCR, Weste blot, and fluorescent determination using Fura 2/AM, respectively. Results: PGF2α (100 nmol/L) significantly increased the cardiomyo-cyte diameter, protein content and [Ca2+]i, and promoted ANP, CaN mRNA, and CaN/NFAT3/GATA4 protein expressions, which were inhibited by either Rb1 in a concentration-dependent manner (50, 100, and 200 μg/mL) or L-arginine (1 mmol/L). NGnitro-L-arginine-methyl ester, a nitric oxide synthase inhibitor, could abolish the effects of L-arginine, but failed to change the effects of Rb1 in the experiments above. Conclusion: The present data implicate that Rb1 attenuates cardiac hypertrophy, the underlying mechanism may be involved in the inhibition of the Ca2+-CaN signal transduction pathway.