The high serum estradiol (E₂) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E₂ concentrations inhibit the activation of nuclear factor kappa B (NF-κB), leading to endometrial epithelial cell (EEC) apoptosis. The objective of this study is to investigate the signaling pathways through which high E₂ results in NF-κB downregulation in EECs. Isolated human EECs were cultured in different concentrations of E₂ (10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷ M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E₂ (10⁻⁷ M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI κB-α) and NF-κB p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs that were exposed in vivo to high serum E₂ from the administration of 20 IU of equine chorionic gonadotropin also demonstrated the same increase in HSF-1 and Hsp70 expression and a decrease in NF-κB. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory κB kinase gamma (IKK-γ) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E₂ concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-γ and inhibits pI κB-α, which consequently suppresses NF-κB activation.