Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.