The eyespot apparatus is an organelle that forms carotenoid-rich globules in diverse flagellated microalgae and functions in phototaxis. The euglenophytes have structurally and functionally distinct eyespot apparatuses from chlorophytes. β-Carotene is the most abundant pigment detected in chlorophytes' eyespots, while xanthophylls such as zeaxanthin and diadinoxanthin have been suggested to function in euglenophytes' eyespots. Here, we investigated the association between carotenoid composition and eyespot formation via pathway-scale mutagenesis using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing in the euglenophyte Euglena gracilis. lycopene cyclase (lcy) mutants exhibited sole lycopene accumulation, defective red eyespots, and phototactic insensitivity. Conversely, β-carotene hydroxylase (cytochrome P450 97h1, cyp97h1) mutants accumulated β-carotene and its hydroxylated products β-cryptoxanthin and zeaxanthin and formed phototactic eyespot apparatuses, while cyp97h1 cyp97f2 double mutants were deficient in β-carotene hydroxylation and mostly lacked functional eyespots. Thus, zeaxanthin is required for the stable formation of functional eyespots in E. gracilis, highlighting evolutionary differences between euglenophytes and chlorophytes in the metabolic regulation of photoreactive organelle formation.