Abstract: Fucoxanthin is one of the common luteins in marine algae, especially in brown algae, which has antibacterial, anti-inflammatory, anti-obesity, anti-diabetes, anti-cancer, regulating intestinal flora, anti-organ fibrosis and other physiological activities. Neoxanthin is a possible precursor of fucoxanthin. In this paper, we constructed a neoxanthin synthesis pathway in Escherichia coli for the first time and the plasmid pTrc99a-crtEBIYZ, which carries five genes (crtE, crtB, crtI, crtY and crtZ) from Pantoea ananatis, and codes for proteins (geranylgeranyl pyrophosphate synthase, phytoene synthase, lycopene cyclase and β-carotene hydroxylase). We transferred the plasmid into E. coli, and obtained the zeaxanthin-producing E. coli BL21 pTrc99a-crtEBIYZ. The yield of zeaxanthin (dry cell mass) was 0.70 mg·g⁻¹. We obtained E. coli pTrc99a-crtEBIYZ pACYCDuet-1-QZEP3 strain by transferring ZEP3 gene from Phaeodactylum tricornutum without signal peptide, and obtained E. coli BL21 NEO successfully by transferring the neoxanthin synthase gene NSY into E. coli BL21 pTrc99a-crtEBIYZ pACYCDuet-1-QZEP3. The concentrations of neoxanthin, violaxanthin and zeaxanthin were 99.27, 150.30 and 119.77 μg·g⁻¹ dry cell mass, respectively.