Abstract: To test whether NtC3H participates in the biosynthesis of flavonoids, and chlorogenic acid (CGA) in tobacco and increases the contents of these secondary metabolites, we constructed the over-expression vector pCXSN::NtC3H and introduced it into Nicotiana tabacum var. samsun via Agrobacterium-mediated transformation. The concentrations of the different secondary metabolites including CGA, rutin, and kaempferol rutinoside in leaves of wild-type and transgenic lines were determined by HPLC. Compared to wild-type samsun tobacco, in transgenic tobacco the concentrations of CGA were increased 3.6-fold, rutin and kaempferol rutinoside were increased 6.1- and 24.6-fold respectively. These results suggested that NtC3H positively regulated the synthesis of flavonoids and CGA in tobacco.