The seed coat of mature black soybean, Glycine max, accumulates a high amount of cyanidin 3-O-glucoside (Cy3G), which is the most abundant anthocyanin in nature. In the pod, it takes two months for the seed coat color change from green to black. However, immature green beans rapidly adopt a black color within one day when the shell is removed. We analyzed the components involved in the color change of the seed coat and detected a new precursor of Cy3G, namely 5,7,3',4'-tetrahydroxyflav-2-en-3-ol 3-O-glucoside (2F3G). Through quantitative analysis using purified and synthetic standard compounds, it was clarified that during this rapid color change, an increase in the Cy3G content was observed along with the corresponding decrease in the 2F3G content. Chemical conversion from 2F3G to Cy3G at pH 5 with air and ferrous ion was observed. Our findings allowed us to propose a new biosynthetic pathway of Cy3G via a colorless glucosylated compound, 2F3G, which was oxidized to give Cy3G.