Objective To establish an oxidative stress model of human valve interstitial cells (VICs) mediated by hydrogen peroxide (H₂ O₂), so as to provide cytology model for research on pathogenesis of valvular heart diseases and screening anti-oxidant drugs. Methods The isolated and cultivated primary human VICs were divided into different groups randomly: control group containing DMEM culture medium with 10% FBS, experimental groups were treated with different concentrations (0, 50, 100, 300, 500, 800, and 1 000 μmol/L) of H₂O₂. H-E staining was used to observe cell morphology, MTT assay was used to estimate cell viability, and Annexin Ⅴ/PI flow cytometer was employed to evaluate the apoptosis of VICs. Results MTT assay showed that the survival rates of VICs were significantly different 24 h after exposure to different concentrations of H₂O₂(P<0.01), with those in 50 and 100 μmol/L groups being significantly higher than that of control group (P<0.05), and the cell survival rate began to decrease with the increase of H₂O₂ concentrations; the decrease became quicker when H₂O₂ concentrations was 800 μmol/L, with a survival rate of (69.8±8.3)%; and the survival rate decreased to (14.3±11.0)% when the concentration reached 1 000 μmol/L. H-E staining showed that at 800 μmol/L, H₂O₂ resulted in crimple and karyopyknosis of the VICs. Flow cytometer results confirmed apparent apoptosis of VICs at the concentration of 800 μmol/L, with the apoptosis already in the middle and advanced stages. Conclusion Oxidative stress model of VICs can be successfully established with H₂O₂, with the optimum concentration of H₂O₂ being 800 μmol/L and the expose period being 24 h.