AIM:To investigate the protective effects of the natural medicinal monomer ecdysterone (ECR) with estrogenic activity against oxidative damage in human lens epithelial cells B3 (HLE-B3) caused by hydrogen peroxide 21(H2O2) and to pursue the possible mitochondrial proteomic regularity of the protective effects.METHODS: HLE-B3 cells were treated with H2O2 (300μmol/L), β-estuarial (E2; 10^-8mol/L) and H2O2, ECR (10^-6mol/L) and H2O2, or left untreated. Altered expression of all mitochondrial proteins was analyzed by protein array and surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS). The mass/charge (M/Z) ratios of each peak were tested by the Kruskal-Wallis rank sum test, and the protein peak value of the M/Z ratio for each treatment by pair comparison was analyzed with the Nemenyi test.RESULTS: H2O2 up-regulated expression of two protein spots (with M/Z of 6 532 and 6 809). When E2 mitigated the oxidative damage, the expression of one protein spot (M/Z 6 532) was down-regulated. In contrast, ECR down-regulated both of protein spots (M/Z 6 532 and 6 809).CONCLUSION: ECR could effectively inhibite H2O2 induced oxidative damage in HLE-B3 cells. The protein spot at M/Z of 6 532 might be the target spot of ECR against oxidative damage induced by H2O2.