The entire flower of Tulipa gesneriana cv. Murasakizuisho is purple, except the bottom, which is blue. To elucidate the mechanism of the different color development in the same petal, we prepared protoplasts from the purple and blue epidermal regions and measured the flavonoid composition by HPLC, the vacuolar pH by a proton-selective microelectrode, and element contents by the inductively coupled plasma (ICP) method. Chemical analyses revealed that the anthocyanin and flavonol compositions in both purple and blue colored protoplasts were the same; delphinidin 3-O-rutinoside (1) and major three flavonol glycosides, manghaslin (2), rutin (3) and mauritianin (4). The vacuolar pH values of the purple and blue protoplasts were 5.5 and 5.6, respectively, without any significant difference. However, the Fe3+ content in the blue protoplast was ∼9.5 mM, which was 25 times higher than that in the purple protoplasts. We could reproduce the purple solution by mixing 1 with two equimolar concentrations of flavonol with λvismax=539 nm, which was identical to that of the purple protoplasts. Furthermore, addition of Fe3+ to the mixture of 1–4 gave the blue solution with λvismax=615 nm identical to that of the blue protoplasts. We have established that Fe3+ is essential for blue color development in the tulip.