Abstract : Background and Aim: TRAIL has sparked a growing interest in oncology due to its ability to selectively trigger cancer cell death while sparing normal cells. The Fas/actin association through ezrin, a member of the ERM protein family, has been reported to regulate early steps of Fas-mediated apoptosis. In this project, we addressed the role of ezrin regarding TRAIL-induced cell death in B lymphoma cell lines, or adherent cancer cell lines (HeLa WT, HCT116, SW480). Methods: Molecular and biochemical approaches were employed to study the relevance of ezrin and its phosphorylation status in TRAIL signaling. Results: We found that ezrin displays a negative function towards TRAIL- and Fas-mediated apoptosis and that the ezrin-mediated TRAIL-induced cell death inhibition led to ezrin activation through phosphorylation/dephosphorylation events at serine 66 and tyrosine 353, but is mainly independent of TRAIL DISC (Death Inducing Signalling Complex) formation or activation. Mutations of these residues to alanine (S66A) or aspartic acid (Y353D) selectively enhanced TRAIL-induced cell death, whereas point mutations mimicking ezrin phosphorylation on S66 (S66D) or a nonphosphorylable variant on Y353 (Y353F) strongly protected cancer cells from apoptosis induced by TRAIL. Moreover, inhibition of the ezrin serine 66 PKA target site, using H89, increased cancer cell sensitivity to TRAIL, while treatment with 8bromocyclic AMP, a PKA activator, decreased TRAIL-induced cell death. In addition, combined TRAIL/cisplatin treatments abrogated ezrin-mediated inhibition of TRAIL-induced apoptosis.