Daidzein has become increasingly popular as a dietary supplement, particularly for postpeak-estrus animals, as a safe and natural alternative estrogen-like compound. However, there is little available safety data of daidzein in laying hens. A study was conducted to examine if high-dose daidzein affected the safety of hens, including mortality, laying performance, egg quality, hematological parameters, clinical chemical parameters, organ development parameters, and hatchability. A total of 2,448 42-wk-old Rugao laying hens were randomly assigned to 4 groups with 6 replicates of 102 birds each (612 laying hens per group). After a 2-wk acclimation period, the birds were fed diets supplemented with 0, 10, 100, or 200 mg/kg of daidzein for 12 wk. The hatchability of setting eggs increased linearly with increasing dietary daidzein supplementation (P = 0.034), while the hatchability of fertile eggs also tended to increase linearly (P = 0.069). The red cell distribution width (RCDW) and coefficient variation of RCDW showed an increasing and then decreasing quadratic response to increasing dietary daidzein supplementation (P = 0.001 and 0.002, respectively). No statistically significant changes were observed in mortality, laying performance, egg quality, clinical chemistry parameters, or organ development parameters (P > 0.05). The magnitude of these hematological changes was such that they were considered to be of no toxicological significance. Therefore, a nominal daidzein concentration of 200 mg/kg is not expected to cause adverse effects following daily administration to laying hens for 84 d.