Sea cucumber is a “healthy” food. Although previous studies have suggested that sea cucumber might serve as a potential rich source of polyunsaturated fatty acids (PUFAs) enriched phospholipid (PL), the molecular species of its PL has rarely been reported. In this study, some 200 glycerophospholipid (GP) species belonging to seven classes in processing by-products (spawns and intestines) of sea cucumber (Stichopus japonicus) were characterized for the first time. Most of the dominant GP species contained PUFAs, especially eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, C20:4n-6). Meanwhile, the lipids contained high levels of PUFA (25.35–45.12% of total FAs) and polar lipid (65.55–85.95% of total lipids) but low levels of cholesterol (0.63–2.26% of total lipids). Among PL, phosphatidylcholine (38.34–65.56 mol%) was dominant. Therefore, PUFA enriched PL in sea cucumber may account for their nutritional and health beneficial effects. Meanwhile, by-products of Stichopus japonicus byproducts provide great potential as health-promoting food ingredients.

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