The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2-Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2-Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.