Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0-41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.