ABSTRACTPhytoene synthase (PSY) is a key enzyme in carotenoid metabolism and often regulated by orange protein. However, few studies have focused on the functional differentiation of the two PSYs and their regulation by protein interaction in the β-carotene-accumulatingDunaliella salinaCCAP 19/18. In this study, we confirmed that DsPSY1 fromD. salinapossessed high PSY catalytic activity, whereas DsPSY2 almost had no activity. Two amino acid residues at positions 144 and 285 responsible for substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Moreover, orange protein fromD. salina(DsOR) could interact with DsPSY1/2. DbPSY fromDunaliellasp. FACHB-847 also had high PSY activity, but DbOR could not interact with DbPSY, which might be one reason why it could not highly accumulate β-carotene. Overexpression ofDsOR, especially the mutantDsORHis, could significantly improve the single-cell carotenoid content and change cell morphology (with larger cell size, bigger plastoglobuli, and fragmented starch granules) ofD. salina. Overall, DsPSY1 played a dominant role in carotenoid biosynthesis inD. salina, and DsOR promoted carotenoid accumulation, especially β-carotene via interacting with DsPSY1/2 and regulating the plastid development. Our study provides a new clue for the regulatory mechanism of carotenoid metabolism inDunaliella.IMPORTANCEPhytoene synthase (PSY) as the key rate-limiting enzyme in carotenoid metabolism can be regulated by various regulators and factors. We found that DsPSY1 played a dominant role in carotenogenesis in the β-carotene-accumulatingDunaliella salina, and two amino acid residues critical in the substrate binding were associated with the functional variance between DsPSY1 and DsPSY2. Orange protein fromD. salina(DsOR) can promote carotenoid accumulation via interacting with DsPSY1/2 and regulating the plastid development, which provides new insights into the molecular mechanism of massive accumulation of β-carotene inD. salina.