AbstractConsidering the potential role of interleukin‐8 (IL‐8) in inflammation, angiogenesis, tumorogenesis, and metastasis, and the involvement of different cell types especially neutrophils and macrophages in those processes, the regulation of IL‐8‐mediated biological responses is important. In this report we provide evidences that oleandrin, a cardiac glycoside potentially inhibited IL‐8‐, formyl peptide (FMLP)‐, EGF‐, or nerve growth factor (NGF)‐, but not IL‐1‐ or TNF‐induced NF‐κB activation in macrophages. Oleandrin inhibited IL‐8‐, but not TNF‐induced NF‐κB‐dependent genes expression. Oleandrin inhibited the binding of IL‐8, EGF, or NGF, but not IL‐1 or TNF. It decreased almost 79% IL‐8 binding without altering affinity towards IL‐8 receptors and this inhibition of IL‐8 binding was observed in isolated membrane. The IL‐8, anti‐IL‐8Rs antibodies, or protease inhibitors were unable to protect oleandrin‐mediated inhibition of IL‐8 binding. Phospholipids significantly protected oleandrin‐mediated inhibition of IL‐8 binding thereby restoring IL‐8‐induced NF‐κB activation. Oleandrin altered the membrane fluidity as detected by microviscosity parameter and a decrease in diphenylhexatriene, a lipid binding fluorophore binding in a dose‐dependent manner. Overall, our results suggest that oleandrin inhibits IL‐8‐mediated biological responses in diverse cell types by modulating IL‐8Rs through altering membrane fluidity and microviscosity. The study might help to regulate IL‐8‐mediated biological responses involved in inflammation, metastasis, and neovascularization. J. Cell. Physiol. 207: 195–207, 2006. © 2005 Wiley‐Liss, Inc.