A previously undescribed acylated anthocyanin was extracted from the red-purple flowers of Pueraria lobata with 5% HOAc-H2O, and determined to be petunidin 3-O-(β-glucopyranoside)-5-O-[6-O-(malonyl)-β-glucopyranoside], by chemical and spectroscopic methods. In addition, two known acylated anthocyanins, delphinidin 3-O-(β-glucopyranoside)-5-O-[6-O-(malonyl)-β-glucopyranoside] and malvidin 3-O-(β-glucopyranoside)-5-O-[6-O-(malonyl)-β-glucopyranoside] were identified. Delphinidin 3,5-di-glucoside, petunidin 3,5-di-glucoside, and malvidin 3,5-di-glucoside, have been known as major components of P. lobata in the former study. However, malonyl esters amounts were detected over 10 times compared with non-malonyl esters amounts. In those anthocyanins the most abundant anthocyanin was petunidin 3-O-(β-glucopyranoside)-5-O-[6-O-(malonyl)-β-glucopyranoside] in total flowers. On the visible absorption spectral curve of fresh red-purple petals, one characteristic absorption maximum was observed at 520 nm, which is similar to those of flowers containing pelargonidin derivatives. In contrast, the absorption spectral curve of old violet petals was observed at 500(sh), 536, 564(sh), and 613(sh) nm, which are similar to those of violet flowers containing delphinidin-type pigments. Pressed juices of both fresh red-purple petals and old violet petals had pH5.2 and 5.5 respectively, and had the same flavonoid constitution. Crude fresh red-purple petal pigments extracted by pH 2.2 and pH 5.2 buffers exhibited the same color and spectral curves as fresh red-purple petals and old violet petals, respectively. Moreover, in a cross-TLC experiment of crude extracted pigments, red-purple color was exhibited by the anthocyanin region and the crossed region of anthocyanins and isoflavone. Thus, it may be assumed that the unusually low pH in the vacuole of fresh petals plays an important role to form red-purple flower color against weak acidic pH in the vacuole of old violet P. lobata petals.