Apoptotic signaling provoked by death receptors, DR4 and DR5, are generally considered to promote cell death and chemosensitivity in multiple cancers, but this view is being thrown into doubt with recent findings that up-regulated DR4 and DR5 in advanced stages of ovarian cancer are associated with the poor prognosis. For this conflict, two reasonable explanations have been proposed: one is that DR4 and DR5 not exclusively mediate apoptotic pathway, but also favor survival signal; another is that apoptotic signals by DR4 and DR5 are disrupted by some regulators. This study identified these two speculations in TRAIL-resistant (SKOV-3ip1 and A2780) or sensitive (OVCAR-3) ovarian cancer cells. Activation of DR4 and DR5 using their specific ligand, TRAIL, activated pro-survival factors including NF-κB, Akt and ERK(1/2) in ovarian cancer SKOV-3ip1 and A2780 cells. Pharmacological inhibition of their activities potentiated TRAIL cytotoxicity, reducing cell viability and increasing apoptosis. Six1, a homeobox transcription factor, had higher expression in SKOV-3ip1 and A2780 cells than in OVCAR-3 cells. Silencing Six1 raised levels of apoptotic factors including cleaved Bid, caspase-8 and caspase-3, and overrode the TRAIL-resistance. Co-treatment with Six1 knockdown and peptidyl O-glycosyltransferase 14 overexpression showed additive effects on apoptosis signal, leading to increased apoptosis in SKOV-3ip1 and A2780 cells. This study demonstrated that pro-survival effects by NF-κB, Akt and ERK(1/2) and anti-apoptosis actions by Six1 disrupt apoptotic functions of TRAIL-Dr4/5 pathway in ovarian cancer, which may explain why up-regulated DR4 and DR5 in ovarian cancer are associated with poor prognosis and low survival ratio of the patients.