Golden-brown xanthophyll fucoxanthin in marine organisms, especially in diatoms, has attracted widespread attention because of its diverse biological activities. However, the biosynthetic pathway of fucoxanthin remains unclear in diatoms. Fucoxanthin may derive from either neoxanthin or diadinoxanthin pathway. However, the key point is whether neoxanthin and its synthesizing genes exist or not. In this study, we successfully identified a few xanthophylls in trace amounts in the concentrated fraction of carotenoids of diatom Phaeodactylum tricornutum cultured at different light intensities with the co-chromatography method, and cloned the neoxanthin synthase (NXS) gene which was not annotated in diatom genome. The NXS knockdown and knockout experiment show a positive correlation in the accumulation of neoxanthin and zeaxanthin while a negative correlation in violaxanthin and fucoxanthin with the expression of NXS. In vitro assay evidenced that neoxanthin is the precursor for fucoxanthin biosynthesis, indicating that other molecules intermediate the conversion between violaxanthin and fucoxanthin. Overall, we cloned a novel gene functioning in neoxanthin biosynthesis, which should aid to clarifying the fucoxanthin biosynthetic pathway in diatom.