Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previously validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl-beta-alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of beta-carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function includes protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.