Secondary metabolites, such as flavonoids, are key participants in biotic and abiotic stress responses in plants. Production of Flavonol Glycosides 3 (PFG3), an R2R3-MYB transcription factor, has been reported to regulate flavonoid biosynthesis and accumulation in Arabidopsis, but its role in drought and other stress responses remains unclear. In this study, we created PFG3-deficient mutants ( pfg3 and pfg3-d1 ) to investigate how PFG3 influences plant flavonoid synthesis and drought/osmotic stress responses. PFG3 was localized in the nucleus and had transcriptional activator activity. The transcript levels of key flavonoid synthesis genes were significantly reduced in pfg3 and pfg3-d1 . PFG3 was expressed mainly in leaves, and its expression was induced by osmotic stress. pfg3 and pfg3-d1 showed impaired accumulation of flavonoids, especially flavonols, and were more sensitive to osmotic and drought stresses. The pfg3 and pfg3-d1 mutants showed less biomass accumulation and a weaker ability to acclimate during drought treatment compared with Col-0 plants. Taken together, our data show that PFG3 is important for plant drought/osmotic stress tolerance by regulating flavonoid biosynthesis. • PFG3 regulates the expression of key flavonoid biosynthesis genes and affects flavonoid accumulation in plants. • PFG3 is expressed mainly in the leaves and is induced by osmotic stress. • PFG3 plays important roles in plant drought/osmotic stress tolerance through the regulation of flavonoid biosynthesis.