Flavonoids are major polyphenol compounds in plants and contribute substantially to the health-promoting benefits of fruit and vegetables. Peach is rich in polyphenols with flavonols as the main flavonoids. To investigate the regulation of flavonol biosynthesis in peach fruit, two R2R3-MYB transcription factor (TF) genes, PpMYB15 and PpMYBF1, were isolated and characterized. Sequence analysis revealed that the PpMYB15 and PpMYBF1 proteins are members of the flavonol clade of the R2R3-MYB family. Real-time quantitative PCR analysis showed that PpMYB15 and PpMYBF1 transcript levels correlated well with the flavonol content and the expression of flavonol synthase (PpFLS1) in different fruit samples. Dual-luciferase assays indicated that both PpMYB15 and PpMYBF1 could trans-activate promoters of flavonoid biosynthesis genes, including chalcone synthase (PpCHS1), chalcone isomerase (PpCHI1), flavanone 3-hydroxylase (PpF3H), and PpFLS1. Transient overexpression of 35S::PpMYB15 or 35S::PpMYBF1 both triggered flavonol biosynthesis but not anthocyanin and proanthocyanidin biosynthesis in tobacco leaves. In transgenic tobacco flowers, overexpression of 35S::PpMYB15 or 35S::PpMYBF1 caused a significant increase in flavonol levels and significantly reduced anthocyanin accumulation, resulting in pale-pink or pure white flowers. These results suggest that PpMYB15 and PpMYBF1 are functional flavonol-specific positive regulators in peach fruit and are important candidates for biotechnological engineering flavonol biosynthesis in plants.