In this study,a new flavonoid-3′,5′-hydroxylase (F3′5′H) gene,named PgF3′5′H (The accession number in GenBank:JQ403611) was isolated from the flower of Platycodon grandiflorus by using RT-PCR and RACE approaches.The full length cDNA sequence of PgF3′5′H is 1 787 bp,with an ORF of 1 599 bp encoded 532 amino acids.Multiple sequence alignment showed that the PgF3′5′H was highly similar with F3′5′H of other plants,and contained several known motifs including “PPGP” motif,I-helix,P450 haem-binding and a special sequence with 9 amino acids liked the Campanula medium F3′5′H.Semi-quantitative RT-PCR analysis showed that the expression level of the PgF3′5′H,with the development of P.grandiflorus flowers and the appearance of anthocyanins in flower tissue,was obviously increased,and got to the maximum in blue buds of the fourth flower developing stages,but absent in leaves.It was speculated that the PgF3′5′H might play an important role in blue anthocyanidin biosynthetic pathway of P.grandiflorus flowers.