Abstract Pathological cardiac hypertrophy induced by increased sympathetic drive can subsequently lead to congestive heart failure, which represents the major cause of morbidity and mortality worldwide. Astragalus polysaccharide (APS) is an active compound extracted from Chinese herb Astragalus membranaceus (AM), a frequently used “Qi-invigorating” herbal medicine in traditional medicine broadly used for the treatment of cardiovascular and other diseases. Currently, little is known about the effect of APS on cardiac hypertrophy. In the present study, we aimed to investigate its effect on cardiac hypertrophy and to clarify its possible mechanisms. In vitro cardiac hypertrophic model induced by isoprenaline (ISO) was employed to explore the anti-hypertrophic action of APS. We found that 10 μM ISO treatment for 48 h caused cultured cardiomyocytes to undergo significant increases in cell surface area, total protein content, protein synthesis as well as the expression of hypertrophic markers, including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which were effectively inhibited by APS in a dose dependent manner. Moreover, we found that APS pretreatment alleviated the augment of intracellular free calcium during cardiac hypertrophy induced by ISO. Our further study revealed that the upregulated expression of calcineurin, translocation of nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) into nucleus and activation of calmodulin kinase II (reflected by p-CaMKII) were dose dependently suppressed by the application of APS. According to this research, APS exerted its anti-hypertrophic action via inhibiting Ca 2+ -mediated calcineurin/NFATc3 and CaMKII signaling cascades, which provided new insights into the application of APS to the therapy of heart diseases.