Abstract: In order to investigate the protective effect of Daidzein（DAI） on H₂O₂-induced senescence of rat cardiomyocytes and its mechanism, we randomly divided the H9 c2 cells into the control group, model group(400 μmol·L^-1 of H₂O₂), and low, medium and high-dose DAI groups(20, 40 and 80 μmol·L^-1 of DAI, respertively). And we observed the cell senescence by senescence-associated β-galactosidase（SA-β-gal） staining. The total antioxidant capacity（T-AOC）, superoxide dismutase（SOD） activity and malondialdehyde（MDA） content were detected by the colorimetric assay. The ROS level was detected by DCFH-DA fluorescence staining. The mitochondrial membrane potential was detected by the Rh123 fluorescence staining. The expressions of p53, p21, kelch-like epichlorohydrin-associated protein-1（Keap1）, nuclear factor E2-associated factor（Nrf2）, heme oxygenase-1（HO-1） proteins in H9 c2 cells were detected with the western blotting method. The results showed that DAI decreased the positive staining of SA-β-gal, down-regulated the expression of p53 and p21 proteins, increased the T-AOC level and SOD activity, decreased the MDA and ROS levels, and maintained the stability of mitochondrial membrane potential in aging H9 c2 cells induced by H₂O₂. DAI inhibited the expression of Keap1 protein, up-regulated Nrf2 and HO-1 protein expression. It is suggested that DAI could delay cellular senescence, enhance cellular antioxidant capacity and inhibit cellular oxidative stress. The mechanism may be related to regulation of Nrf2/HO-1 signaling pathways.