The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.