A full-length cDNA of a putative flavonoid 3'-hydroxylase (F3'H) gene encoding a key enzyme in the production of cyanidin was cloned from a lisianthus (Eustoma grandiflorum) petal. Lisianthus F3'H (EgF3'H) shares 75.1, 73.8, and 68.2% amino acid identity with Arabidopsis thaliana, Ipomoea nil, and Petunia hybrida, respectively. RT-PCR revealed that wild-type lisianthus flowers accumulated higher levels of F3'H mRNA during the early stages of development than in the late stages. The accumulated F3'H transcript levels in leaves were similar to those in flowers in the early stages of development. Overexpression of lisianthus F3'H cDNA altered flower color from red to blue in the I. nil cultivar ‘Violet’, which lacks a functional F3'H gene. In addition, the transgenic ‘Violet’ plants accumulated cyanidin and peonidin at similar levels to wild-type I. nil. Taking these findings together, this study demonstrates that EgF3'H functions as a flavonoid 3'-hydroxylase with a role in the synthesis of cyanidin and peonidin pigments.