Backgroud <p>As one kind of flavonoids in plants, anthocyanins are the main important pigments, which are responsible for painting flowers and fruits with various color to facilitate seed distribution and fertilization. The biosynthesis of anthocyanins is regulated by a battery of structural genes and transcription factors. However, the molecular mechanisms underlying anthocyanins accumulation in <i>Rhododendron agastum</i> are still unclear.</p> Results <p>Here, the transcriptome and metabolite analysis were compared to reveal candidate genes for flower color formation in <i>R. agastum</i> (pink colors) through RNA-Seq and HPLC-MS/MS. A total of 33 structural genes and 11 R2R3 MYBs were predicted as anthocyanins biosynthesis-related genes. Then, one critical structural gene, <i>Dihydroflavonol 4-reductase 2</i> (<i>RaDFR2</i>), was isolated and characterized in vitro as well as in vivo to test its function on anthocyanins biosynthesis. The results showed that RaDFR2 had catalytic activity towards dihydroquercetin (DHQ) and dihydromyricetin (DHM), could restore anthocyanins biosynthesis of <i>Arabidopsis tt3</i> mutant, and lead to enhanced anthocyanins accumulation in transgenic tobacco by up-regulating the expression levels of structural genes and regulatory genes.</p> Conclusions <p>This work lays the foundation for revealing the molecular mechanism of flower color formation in <i>R. agastum</i>, and provides genetic resources for improving outward appeal and quality of <i>R. agastum</i> through molecular breeding.</p>