Flavonol synthase (FLS) is a key enzyme of the flavonoid biosynthetic pathway, which catalyzes the conversion of dihydroflavonols into flavonols. In this study, the FLS geneIbFLS1was cloned and characterized from sweet potato. The resulting IbFLS1 protein showed a high similarity with other plant FLSs. The conserved amino acids (HxDxnH motifs) binding ferrous iron and residues (RxS motifs) binding 2-oxoglutarate were found in IbFLS1 at conserved positions, as in other FLSs, suggesting that IbFLS1 belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. qRT-PCR analysis showed an organ-specific pattern of expression of theIbFLS1gene, which was predominantly expressed in young leaves. The recombinant IbFLS1 protein could catalyze the conversion of dihydrokaempferol and dihydroquercetin to kaempferol and quercetin, respectively. The results of subcellular localization studies indicated that IbFLS1 was found mainly in the nucleus and cytomembrane. Furthermore, silencing theIbFLSgene in sweet potato changed the color of the leaves to purple, substantially inhibiting the expression ofIbFLS1and upregulating the expression of genes involved in the downstream pathway of anthocyanin biosynthesis (i.e.,DFR,ANS, andUFGT). The total anthocyanin content in the leaves of the transgenic plants was dramatically increased, whereas the total flavonol content was significantly reduced. Thus, we conclude thatIbFLS1is involved in the flavonol biosynthetic pathway and is a potential candidate gene of color modification in sweet potato.