Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. However, sensitivity of cancer cells for induction of apoptosis by TRAIL varies considerably. Therefore, it is important to develop agents that overcome this resistance. We show, for the first time, that eupatolide, the sesquiterpene lactone isolated from the medicinal plant Inula britannica, sensitizes human breast cancer cells to TRAIL-induced apoptosis. Treatment with TRAIL in combination with subtoxic concentrations of eupatolide enhanced the TRAIL-induced cytotoxicity in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells, whereas each reagent alone slightly induced cell death. The combination induced sub-G1 phase DNA content and annexin V-staining in MCF-7 cells, which are major features of apoptosis. Apoptotic characteristics induced by the combined treatment were significantly inhibited by a pan-caspase inhibitor. The sensitization to TRAIL-induced apoptosis was accompanied by the activation of caspase-8 and was concomitant with Bid and poly(ADP-ribose) polymerase (PARP) cleavage. Treatment of eupatolide alone significantly down-regulated the expression of cellular FLICE inhibitory protein (c-FLIP) in MCF-7 cells. Furthermore, enforced expression of c-FLIP significantly attenuated the apoptosis induced by this combination in MCF-7 cells, suggesting a key role for c-FLIP down-regulation in these events. We also observed that euaptolide inhibited AKT phosphorylation in a dose- and time-dependent manner. Moreover, inhibition of Akt by LY294002, a specific PI3K inhibitor, down-regulated c-FLIP expression in MCF-7 cells. Taken together, these results indicate that eupatolide could augment TRAIL-induced apoptosis in human breast cancer cells by down-regulating c-FLIP expression through the inhibition of AKT phosphorylation and be a valuable compound to overcome TRAIL resistance in breast cancer cells.