The cytotoxicity and apoptotic effect of phytoceramide Ⅳ (N-palmitoyl-phytospingosine) were investigated by preparaing the ceramide containing liposomes using cholesteryl hemisuccinate (CHEMS) and methoxypolyethylene glycol (2000) cholesteryl succinate (PEGCHS). The ceramide-containing liposomes prepared using ultrasonication contained CHEMS and the phytoceramide Ⅳ at a molar ratio of 1:1. Gel chromatography and high performance liquid chromatography equipped with an evaporative light-scattering detection (HPLC-ELSD) were used to analyze the liposomes. The results show that the ceramide entrapment efficiency is over 90% and the molar ratio of phytoceramide Ⅳ to CHEMS is 1:1.4. The ratio of ceramide to CHEMS as well as the ultrasonication duration affects the liposome properties. The phytoceramide Ⅳ encapsulated in the liposomes reduces the cellular activity of the murine mastocytoma cell line P815 in a dose-dependent manner and the reduction of cellular activity is due to cell apoptosis. 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining further supports this result. The encapsulation of highly hydrophobic ceramides into liposomal formulations could become a potential candidate for enhanced apoptosis.