Flavonoids are secondary metabolites that contribute substantially to the quality of Ginkgo biloba. Plant flavonoid accumulation is controlled by transcriptional regulation of the genes that encode the biosynthetic enzymes, in which the R2R3-MYB transcription factor is a key factor. In this study, we describe the cloning and functional characterization of a R2R3-MYB transcription factor gene, GbMYBF2, isolated from G. biloba. GbMYBF2 encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Comparative and bioinformatics analyses showed that GbMYBF2 is more closely related to the repressor R2R3-MYB subfamily involved in flavonoid biosynthesis. Tissue expression pattern analysis showed that GbMYBF2 was constitutively expressed in leaves, fruits, stems, and roots, wherein the level of transcription in the roots is significantly higher than that in the stems, leaves, and fruits. During G. biloba leaf growth, the transcription of GbMYBF2 is negatively correlated with the flavonoid content, suggesting that the GbMYBF2 gene is responsible for the repressed flavonoid biosynthesis. Transgenic Arabidopsis plants that overexpress GbMYBF2 exhibit an inhibition of flavonoid and anthocyanin biosynthesis compared with the untransformed Arabidopsis plants. In addition, the overexpression of GbMYBF2 in Arabidopsis clearly downregulates the expression of the structural genes that control the synthesis of flavonoids and anthocyanins. These findings suggest that GbMYBF2 may have a key role in repressing transcription in regulating the biosynthesis of flavonoids in G. biloba.