Dihydroflavonol 4-reductase (DFR), a key regulatory enzyme, participated in the biosynthesis of anthocyanins, proanthocyanidins and other flavonoids that essential for plant survival and human health. However, the role of this enzyme in is still unknown. Here, three putative genes were firstly isolated from . Phylogenetic analysis indicated that was classified into DFR subgroup, while the rest two were clustered into other NADPH-dependent reductases. Then, functions of the three genes were further characterized. Expression analysis showed that transcripts had strong correlations with the accumulation pattern of anthocyanin during the flower developmental, whereas other two were not, this suggested the potential roles of in anthocyanin biosynthesis. Subsequently, all three clones were functionally expressed in , but confirming that only encode active DFR proteins that catalyzed the reduction of dihydroflavonols to leucoanthocyanidin. Consistant with the biochemical assay results, overexpressing in Arabidopsis mutant successfully restored the deficiency of anthocyanin and proanthocyanidin, hinting its function as DFR in planta. Additionally, heterologous expression of in transgenic tobacco contributed to darker flower color up-regulating the expressions of endogenous and , which suggested that was involved in flower color development. In summary, this study validates the functions of and expands our understanding of anthocyanin biosynthesis in .