Cisapride has been shown to have electrophysiological effects on the heart. The aim of this study was to investigate whether cisapride has effects on cardiac hypertrophy. Rat and cellular models of cardiac hypertrophy were used in this study. Cell surface area (CSA), mRNA and protein expression were used to evaluate cardiac hypertrophy. Cardiac function was measured by echocardiography. Cisapride attenuated ISO-induced increase in CSA in a dose-dependent manner in cultured neonatal rat cardiomyocytes. A significant anti-hypertrophic effect was achieved by cisapride 0.01μM (P<0.05). Cisapride repressed the increased mRNA levels of ANP, BNP, β-MHC in ISO-treated cells (P<0.05). However, mallotoxin or GR113808 did not influence anti-hypertrophic effects of cisapride. In addition, cisapride inhibited the increase of intracellular Ca(2+) ([Ca(2+)]i) and the upregulation of protein levels of calcineurin and NFATc-3 (P<0.05) as well as prevented the downregulation of p-NFATc-3 (P<0.01) induced by ISO. Consistently, cisapride (0.5mg/kg/day) produced inhibitory effects on cardiac hypertrophy, including the suppression of ANP, BNP, β-MHC, calcineurin, and NFATc-3; elevation of p-NFATc-3; reduction of cross-sectional area of cardiomyocytes in rat heart; and restoration of cardiac dysfunction by improving left ventricular diastolic and systolic performance. Importantly, cisapride 0.5 and 5.0mg/kg/day did not cause prolongation of QT and QTc intervals in rats. In conclusion, cisapride possesses a prominent anti-hypertrophic property which is likely to be conferred by its ability to downregulate Ca(2+)/calcineurin/NFAT and the present data provide new insight into this drug action.