Buckwheat (Fagopyrum esculentum) contains high amounts of flavonoids, especially flavonols (e.g., rutin), which are thought to be highly beneficial for human health. Little is known, however, about the regulation of flavonol synthesis in buckwheat. We identified a buckwheat gene encoding an R2R3 MYB transcription factor, and named this gene FeMYBF1. Analysis of the deduced amino acid sequence and phylogenetic analysis suggested that FeMYBF1 encodes an ortholog of the Arabidopsis flavonol regulators AtMYB11, AtMYB12 and AtMYB111. Expression of FeMYBF1 in a flavonol-deficient Arabidopsis triple mutant (myb11 myb12 myb111) restored flavonol synthesis. Constitutive expression of FeMYBF1 driven by the CaMV 35S promoter in Arabidopsis resulted in over-accumulation of flavonol glycosides and upregulation of the expression of AtFLS1. Transient expression assays showed that FeMYBF1 activated the promoter of the Arabidopsis gene encoding AtFLS1, and the promoters of buckwheat genes related to anthocyanin and proanthocyanidin synthesis such as dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) in addition to genes encoding FLS. The results indicate that FeMYBF1 regulates flavonol synthesis and may have a role in synthesis of other flavonoid compounds, and also that buckwheat may have alternative pathway of flavonol synthesis through DFR and LDOX.