Tisochrysis lutea (T. lutea, ex T. Isochrysis galbana or T-Iso) is a marine haptophyte that was first isolated from Tahiti seawater. Because of its high content in lipids, this tropical species is commonly used in aquaculture to feed fishes, crustaceans and molluscs larvae. It is also a rich source of fucoxanthin with a high potential for nutraceutical, cosmetic and pharmaceutical applications. The purpose of the present study was to detail the pigment composition of T. lutea and to develop an efficient process to recover highly purified fucoxanthin. Using ultra performant liquid chromatography coupled to diode arrays and high-resolution mass spectrometry detectors (UPLC-DAD-MS/MS), we demonstrated for the first time the presence of echinenone, 3-hydroxy-echinenone and chlorophyll c2-monogalactosyldiacylglycerol [18:4/14:0] in unstressed cultures of T. lutea. The chemotaxonomic relevance of this updated pigment composition was discussed in relation to the Haptophyta phylum. A two-step purification of fucoxanthin was then optimized using centrifugal partition chromatography coupled to flash chromatography. This process allowed the efficient isolation of fucoxanthin (purity > 99%), that was further assessed as a low-toxicity antineoplastic and chemosensitizing natural product in human chemoresistant melanoma cells. This carotenoid exerted an antiproliferative activity in A2058 melanoma cells and reversed in vitro their chemoresistance to dacarbazine, a DNA-alkylating agent clinically used for the treatment of metastatic melanoma.