Chimeric repressor gene-silencing technology (CRES-T) is an efficient gene suppression system in a wide variety of dicots and monocots. In this study, we demonstrated that the CRES-T system functions in Japanese gentian. A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue-flowered gentian. Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal. HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type, suggesting competitive accumulation of these two types of compounds. The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F3′5′H, DFR and ANS, were strongly suppressed in petals of these transgenic plants. In contrast, the ‘early’ flavonoid biosynthetic genes, such as CHS and FNSII, were not affected. Since FNSII is expressed more strongly in the lower part of petals than in the upper part, the absence of pigmentation in the lower parts might be induced by flavone synthesis. These results demonstrated that the suppression of anthocyanin biosynthetic genes by CRES-T was successfully applied to Japanese gentian to change petal color; therefore, this system could be useful for generating novel flower colors and patterns. Transgenic plants produced in this study might be utilized as elite materials in the breeding of Japanese gentian in the near future.