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Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass.