Objective: To establish a cardiomyocyte hypertro-phy model, observe the effects of rutin on hypertrophic cardiomyocytes, and explore the possible mechanism. Methods: Cardiomyocytes of neonatal rats were cultured in vitro. The survival rate of cardiomyocytes was observed by CCK-8 method. The surface area of myocardial cells and the concentration of intracellular calcium ions were detected by laser confocal microscopy. The activity of Ca2+ ATPase was determined by the enzymatic reaction of broken cells. The expressions of Ca MKII, HDAC, c-Jun, ANP, BNP, β-MHC, Ca N, and NFAT-3 proteins were detected by western blot. The concentration of nitric oxide (NO) and the activity of NOS were determined by colorimetry. Results: Different concentrations of rutin could in-hibit the decrease of survival rate of cardiomyocytes induced by Angiotensin II (Ang II), the increase of the surface area, the increase of the Ca2+ concentration in cardiomyocytes, and the decrease of the activity of Ca2+ ATPase, the increment of the relative expressions of Ca MKII, HDAC, Ca N, and NFAT-3 proteins, and the increase of the relative expressions of c-Jun, ANP, BNP, and β-MHC proteins. The decrease in NO concentration and NOS activity also has been inhibited to a certain degree. Conclusion: Rutin has significant inhibitory and protective effects on Ang II-induced hypertrophic cardiomyocytes, and the mechanism may be related with the release of NO, the regulation of intracellular Ca2+ concentration and Ca2+ ATPase activity, as well as the block of calcium ion-mediated Ca N-NFAT-3 and the Ca MK II-HDAC signal transduction pathways. © 2019 Science Printers and Publishers.