Objective:To observe the effect of polydatin on hypertrophy of rat myocardial cells and proliferation of fibroblasts in vitro, and to explore its antioxidation effect. MethodsLeft ventricle of neonatal SD rats were selected and its myocardial cells and fibroblasts were isolated and cultured by differential attachment method. The cardiomycytes and fibroblasts were divided into 5 groups:normal control group, polydatin control group (10-4 mol/L), model group (10-6 mol/L ISO), and low concentration polydatin group (10-5 mol/L+ISO 10-6 mol/L) and high concentration polydatin group (10-4 mol/L +ISO 10-6 mol/L). Different drugs intervened for 2 days, and protein content of cardiomycytes was determined by Coomassie Brilliant Blue; activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected; proliferation of fibroblasts and concentration of SOD, GSH-Px and NO in cell culture medium were determined.  Results:Compared with the normal control group, protein content of cardiomyocytes was increased in the model group, and activities of SOD and GSH-Px were reduced markedly (P<0.05); compared with the model group, the protein content in high concentration polydatin group was markedly reduced, while the activities of SOD and GSH-Px were significantly increased (P<0.05); GSH-Px activity of the high concentration polydatin group was superior to that of the low concentration polydatin group, with signficiant difference between the two groups (P<0.05). Compared with the normal control group, 10-6 mol/L ISO could induce fibroblast proliferation and reduce the NO content, and activities of SOD and GSH-Px (P<0.05); compared with the model group, proliferation of fibroblasts was inhibited in the high concentration polydatin group, and the NO content, and activities of SOD and GSH-Px were increased markedly (P<0.05).  Conclusion: Polydatin shows antagonism on ISO-induced hypertrophy of neonatal cardiomyocytes and proliferation of fibroblasts, its mechanism effect may relate to antioxidant and the production of NO.