The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a semi-dominant, single-locus mutation. It induces the accumulation of high levels of beta-carotene in various tissues that are normally devoid of carotenoids, turning them orange. Using a map-based cloning strategy, we identified a single candidate gene representing Or and successfully verified its identity by phenotypic complementation in the wild type cauliflower. The Or gene encodes a plastid membrane protein containing the DnaJ zinc figure domain. A likely gain-of-function mutation from a 4.3-kb retrotransposon insertion in the Or allele confers the orange phenotype in the mutant. Southern blot analysis revealed that Or is a single-copy sequence in the cauliflower genome. High level of expression of the Or gene and the protein was found in very young leaves, curds, and flowers at comparable abundance between wild type and the Or mutant. Or likely functions in regulating the differentiation of some non-photosynthetic plastids into chromoplasts, which provide the deposition “sink” for carotenoid accumulation. Or orthologs with high degree of sequence homology were found in many other higher plants, including Arabidopsis, tomato, maize, and rice. Successful phenotypic complementation of Or in Arabidopsis provides strong evidence that Or can work across species to enhance carotenoid accumulation in other important crops.