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The xanthophyll cycle is a photoprotective mechanism in plants and algae, which protects the photosynthetic system from excess light damage under abiotic stress. Zeaxanthin is considered to play a pivotal role in this process. In this study, the relative content of xanthophylls was determined using HPLC-MS/MS in <i>Ulva prolifera</i> exposed to different salinities. The results showed that high-salt stress significantly increased the relative content of xanthophylls and led to the accumulation of zeaxanthin. It was speculated that the accumulated zeaxanthin may contribute to the response of <i>U. prolifera</i> to high-salt stress. Zeaxanthin epoxidase (<i>ZEP</i>) is a key enzyme in the xanthophyll cycle and is also involved in the synthesis of abscisic acid and carotenoids. In order to explore the biological function of <i>ZEP</i>, a <i>ZEP</i> gene was cloned and identified from <i>U. prolifera</i>. The CDS of <i>UpZEP</i> is 1122 bp and encodes 373 amino acids. Phylogenetic analysis showed that <i>UpZEP</i> clusters within a clade of green algae. The results of qRT-PCR showed that high-salt stress induced the expression of <i>UpZEP</i>. In addition, heterologous overexpression of the <i>UpZEP</i> gene in yeast and <i>Chlamydomonas reinhardtii</i> improved the salt tolerance of transgenic organisms. In conclusion, the <i>UpZEP</i> gene may be involved in the response of <i>U. prolifera</i> to high-salt stress and can improve the high-salt tolerance of transgenic organisms.