R2R3-MYB transcription factors are known to play important regulation role in the flavonoid biosynthesis of plants. In this study, a gene FtMYB1 belonging to the R2R3-MYB gene family was cloned from Tartary buckwheat cultivar ‘Chuanqiao No.1’.The FtMYB1 transcription factor was found to exhibit the transcriptional activation activity in yeast, and its expression of FtMYB1 gene was induced by Methyl jasmonate (MeJA) and abscisic acid (ABA) treatment. Overexpressing the FtMYB1 gene in Tartary buckwheat hairy root showed a significant reduction in contents of total flavonoid and rutin if compared with wild type. Furthermore,the expression of FLS and RT1 genes, which encode for flavonol synthetase (FLS) and rhamnosyltransferase (RT) in Tartary buckwheat, respectively, were significantly lower in the FtMYB1 overexpressing hairy roots than that of wild type. Taken together, our results suggested that FtMYB1 may function in inhibition of the flavonol biosynthesis by repressing the expression of FLS and RT1 genes.