<jats:title>ABSTRACT</jats:title> <jats:p> Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of <jats:named-content content-type="genus-species">Nostoc</jats:named-content> sp. strain PCC 7120, <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic> using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13 <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cjs0808.jpeg" /> C-14, C-15 <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cjs0808.jpeg" /> C-15′, and C-13′ <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cjs0808.jpeg" /> C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic> .