In this study, the pathway of b-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates b-citraurin predominantly, and Miyagawa-wase, which does not accumulate b-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of b-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of b-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of b-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved b-cryptoxanthin and zeaxanthin at the 7,8 or 79,89 position. But other carotenoids tested in this study (lycopene, a-carotene, b-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of b-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of b-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of b-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.