This experiment investigated the antioxidant effects of equol on oxidative stress induced by H2O2 in chicken intestinal epithelial cells (IEC). IEC, from Lingnan yellow broiler chick embryos at embryonic day 18, were cultured in Dulbecco's modified Eagle's medium/F12. Cells were pretreated with 0, 10, 100, or 500 nM equol for 24 h before exposure to 300 μM H2O2 during a further 24 h. Oxidative damage was assessed by photomicrographs of cells, measuring cell proliferation, malondialdehyde (MDA) content, and antioxidative capacity from cellular total superoxide dismutase (T-SOD) activity, as well as the relative expressions of Nrf2, Bcl-2, SOD-1, GSH-Px3, Claudin-1. Treatment with 300 μM H2O2 caused serious damage to cells, with fewer normal intestinal epithelial cells, revealed by photomicroscopy. Treatment with 300 μM H2O2 significantly decreased live cell numbers compared with controls and prior treatment with equol had no effect in offsetting this action of H2O2 (P > 0.05). Compared with the cells treated just with H2O2, pre-treatment with 10, 100 and 500 nM equol significantly enhanced T-SOD activity (P < 0.05), while 10 and 100 nM equol before H2O2 significantly enhanced T-SOD activity compared with the untreated controls (P < 0.05). In cells pre-treated with 100 nM equol, the relative abundance of Nrf2 transcripts increased from the controls (P < 0.05) but expressions of Bcl-2, GSH-Px3, or SOD-1 were unaffected (P > 0.05). Pre-treatment with 10 and 100 nM equol significantly increased the transcript abundance of Claudin-1 (P < 0.05). Equol is shown here to protect IECs from oxidative damage by promoting the expression of antioxidant genes, increasing the activities of antioxidant enzymes, and by enhancing antioxidant capacity; 100 nM equol appeared to be the most effective concentration.