Cancer cells are known to release extracellular vesicles that often promote disease development and progression. The present study investigated protein content and glycosylation pattern of ectosomes released in vitro by human primary uveal melanoma Mel202 cell line. Ectosomes released by Mel202 cells were isolated from conditioned media by sequential centrifugation and nanoLC-MS/MS approach was used to determine their protein content. Subsequently, proteins from ectosomes, the whole cell extracts and the membrane fractions were probed with a panel of lectins to reveal characteristic glycan structures. As many as 2529 unique proteins were identified, and many of them are known to be involved in cancer cell proliferation and altered metabolism, tumor invasion, metastasis or drug resistance. Lectin-based studies showed a distinct glycosylation pattern between Mel202 ectosomes and parental cell membranes. Selective enrichment of ectosomal proteins with bisected complex type N-glycans and α2,6-linked sialic acid may be significant for ectosome formation and sequestration. Our data supports recent findings that ectosomes derive from particular regions of the cell membrane and thus contain a unique protein and glycan composition. Exploring whether cancer-related proteins and glycans are enriched in ectosomes isolated from body fluids of cancer patients should became a subject of future in-depth studies.