Background: TLR4/MD-2 complex-mediated MyD88-dependent activation of NF-kappa B and Akt promotes tumor-associated immunosuppression in epithelial ovarian cancer (EOC) via induction of immunesuppressive cytokines and indoleamine 2,3-dioxygenase (IDO). Atractylenolide I (AO-1) is a naturally occurring sesquiterpene lactone known to change the conformational ensemble of human MD-2 on EOC cells. This study examined the modulation by AO-1 of TLR4/MD-2 complex-mediated MyD88/NF-kappa B signaling. Methods: The expression and activation of NF-kappa B, Akt and IDO1 by MyD88+EOC SKOV3 cells was determined using western blot; the TLR4/MD-2 complex on SKOV3 cells and the phenotype of T lymphocytes were determined using flow cytometry; IDO activity was evaluated by measuring l-kynurenine; Immunesuppressive cytokines were detected using ELISA; T-cell proliferation to mitogen stimulation was assessed by MTT assay; the cytotoxicity of lymphocytes and NK cells was measured using LDH-cytotoxicity assay. Results: AO-1 could down-regulate expression of TLR4/MD-2 complex, resulting in downregulation of MyD88/NF-kappa B signaling and activation of NF-kappa B, Akt and IDO1 and secretion of IL-6, TGF-beta 1, VEGF and IL-17A by EOC SKOV3 cells, and further reduce increased levels of regulatory T cells (Treg cells) and improve decreased proliferative response and antitumor cytotoxicity of T lymphocytes exposed to EOC SKOV3 cell supernatant. Conclusion: AO-1 may reverse EOC cell-mediated immunosuppression through blocking TLR4/MD-2 complex-mediated MyD88/NF-kappa B signaling.