Abstract: To investigate the function and mechanism of β-carotene hydroxylase (CHYB) in Chlamydomonas reinhardtii, CRISPR/Cas9-mediated gene editing was used to precisely knockout CHYB gene from Chlamydomonas reinhardtii and successfully isolated the chyb mutants. Phenotypic analysis revealed that CHYB knockout significantly decreased photosynthetic activity of photosystem II (PSII), light utilization efficiency (Alpha), and photosynthetic electron transport rate (ETR_max) in chyb mutants, ultimately reducing cell growth rate. Under high light conditions, the photoprotection related non-photochemical quenching ability of chyb mutants was inhibited. Targeted metabolomics analysis revealed the contents of zeaxanthin, antheraxanthin, and violaxanthin, which were the key components of the xanthophyll cycle, were significantly reduced in chyb mutants, thereby affecting the photoprotection ability of Chlamydomonas reinhardtii. In addition, mutations in the CHYB gene were found to affect flagellar beating coordination, resulting in the inability of Chlamydomonas reinhardtii to sustain linear movement. These findings establish dual physiological roles of CHYB in Chlamydomonas reinhardtii: (1) maintaining the non-photochemical quenching ability of Chlamydomonas reinhardtii by regulating the synthesis of key components of xanthophyll cycle; (2) potentially regulating flagellar beating coordination via carotenoid synthesis in eyespots. These findings provide novel insights into the role of carotenoids in regulating the environmental adaptability of photosynthetic organisms.