The fluidity of neuronal membranes plays a pivotal role in brain aging and neurodegeneration. In this study, we investigated the role of the omega-3 fatty acid docosahexaenoic acid (DHA) in modulation of membrane fluidity, APP processing, and protection from cytotoxic stress. To this end, we applied unilamellar transfer liposomes, which provided protection from oxidation and effective incorporation of DHA into cell membranes. Liposomes transferring docosanoic acid (DA), the completely saturated form of DHA, to the cell cultures served as controls. In HEK-APP cells, DHA significantly increased membrane fluidity and non-amyloidogenic processing of APP, leading to enhanced secretion of sAPPα. This enhanced secretion of sAPPα was associated with substantial protection against apoptosis induced by ER Ca(2+) store depletion. sAPPα-containing supernatants obtained from HEK-APP cells exerted similar protective effects as DHA in neuronal PC12 cells and HEK293 control cells. Correlating to further increased sAPPα levels, supernatants obtained from DHA-treated HEK-APP cells enhanced protection, whereas supernatants obtained from DHA-treated HEK293 control cells did not inhibit apoptosis, likely due to the low expression of endogenous APP and negligible sAPPα secretion in these cells. Further experiments with the small molecule inhibitors LY294002 and SP600125 indicated that sAPPα-induced cytoprotection relied on activation of the anti-apoptotic PI3K/Akt pathway and inhibition of the stress-triggered JNK signaling pathway in PC12 cells. Our data suggest that liposomal DHA is able to restore or maintain physiological membrane properties, which are required for neuroprotective sAPPα secretion and autocrine modulation of neuronal survival.