O-Methyltransferases, which are involved in methylation of secondary metabolites, mediate the methylation of small molecules such as flavonoids, alkaloids, and plant hormones. Among them, flavonoids are an important class of secondary metabolites in plant and exhibit a wide spectrum of pharmacological properties. Modification of flavonoids extend and change pharmaceutical characterization of them as well as the structure. Populus deltoids caffeoyl CoA OMT gene (PopCCoAOMT) was amplified by RT-PCR, cloned and characterized. PopCCoAOMT had a homology of 94% and 90.8% with caffeoyl CoA OMTs of Nicotiana tabacum and Arabidopsis thaliana, and encoded 28.0 kDa in molecular weight. The reaction product of PopCCoAOMT enzyme methylated caffeoyl-CoA, quercetin, and luteolin to feruoyl-CoA, isorhamnetin, and chrisoeryol, respectively. PopCCoAOMT enzyme was shown to be active with compounds associated with substrates having an ortho hydroxyl group and a double bond at between 2 and 3 position of carbon.