Vitexin is a flavone glycoside isolated from the leaf of Crataeguspinnatifida Bunge, the utility of which has been demonstrated in several cardiovascular diseases. However, its role in cardiac hypertrophy remains unclear. In the present study, we aimed to determine whether vitexin prevents cardiac hypertrophy induced by isoproterenol (ISO) in cultured neonatal rat ventricular myocytes in vitro and pressure overload-induced cardiac hypertrophy in mice in vivo. The results revealed that vitexin (10, 30, and 100 μM) dose-dependently attenuated cardiac hypertrophy induced by ISO in vitro. Furthermore, vitexin (3, 10, and 30 mg kg(-1)) prevented cardiac hypertrophy induced by transverse aortic constriction as assessed by heart weight/body weight, left ventricular weight/body weight and lung weight/body weight ratios, cardiomyocyte cross-sectional area, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that vitexin inhibited the increment of the resting intracellular free calcium induced by ISO. Vitexin also inhibited the expression of calcium downstream effectors calcineurin-NFATc3 and phosphorylated calmodulin kinase II (CaMKII) both in vitro and in vivo. Taken together, our results indicate that vitexin has the potential to protect against cardiac hypertrophy through Ca2+-mediated calcineurin-NFATc3 and CaMKII signaling pathways.