Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant.