Dietary supplementation with eicosapentaenoic acid (EPA) improves the prognosis of chronic inflammatory diseases, including atherosclerosis. The mechanism underlying these beneficial effects, however, remains to be elucidated. Here we show that EPA protects endothelial cells from anoikis through upregulation of the cellular FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme)-inhibitory protein (cFLIP), an endogenous inhibitor of caspase-8. EPA-induced upregulation of cFLIP expression was partially suppressed by the phosphatidylinositol-3-kinase inhibitor wortmannin. Conversely, treatment with insulinlike growth factor-1 (IGF-1), an activator of phosphatidylinositol-3-kinase/Akt signaling, or infection with an adenoviral construct expressing the constitutively active Akt gene induced upregulation of cFLIP expression. In addition, pretreatment of endothelial cells with either EPA or IGF-1 protected them from anoikis, suggesting that EPA-induced protection against anoikis is partially mediated through activation of Akt. On the other hand, when endothelial cells were already detached, treatment of these cells with EPA but not with IGF-1 protected them against anoikis. Importantly, EPA restored cFLIP expression without activating Akt signaling in detached endothelial cells, whereas IGF-1 had no effect. Additionally, exogenously restored expression of cFLIP by the tetracyclineregulated adenovirus system protected endothelial cells against anoikis. Furthermore, EPA was protective against the loss of endothelium in an organ culture of rat aortas. These findings suggest that EPA protects against endothelial cell anoikis through restoration of cFLIP expression, which might contribute to the mechanism underlying the beneficial effects of EPA in patients with hypertension.